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Sample records for 2d fluorescence spectroscopy

  1. Rapid-scan coherent 2D fluorescence spectroscopy.

    PubMed

    Draeger, Simon; Roeding, Sebastian; Brixner, Tobias

    2017-02-20

    We developed pulse-shaper-assisted coherent two-dimensional (2D) electronic spectroscopy in liquids using fluorescence detection. A customized pulse shaper facilitates shot-to-shot modulation at 1 kHz and is employed for rapid scanning over all time delays. A full 2D spectrum with 15 × 15 pixels is obtained in approximately 6 s of measurement time (plus further averaging if needed). Coherent information is extracted from the incoherent fluorescence signal via 27-step phase cycling. We exemplify the technique on cresyl violet in ethanol and recover literature-known oscillations as a function of population time. Signal-to-noise behavior is analyzed as a function of the amount of averaging. Rapid scanning provides a 2D spectrum with a root-mean-square error of < 0.05 after 1 min of measurement time.

  2. Quantum process tomography by 2D fluorescence spectroscopy

    SciTech Connect

    Pachón, Leonardo A.; Marcus, Andrew H.; Aspuru-Guzik, Alán

    2015-06-07

    Reconstruction of the dynamics (quantum process tomography) of the single-exciton manifold in energy transfer systems is proposed here on the basis of two-dimensional fluorescence spectroscopy (2D-FS) with phase-modulation. The quantum-process-tomography protocol introduced here benefits from, e.g., the sensitivity enhancement ascribed to 2D-FS. Although the isotropically averaged spectroscopic signals depend on the quantum yield parameter Γ of the doubly excited-exciton manifold, it is shown that the reconstruction of the dynamics is insensitive to this parameter. Applications to foundational and applied problems, as well as further extensions, are discussed.

  3. Solution conformation of 2-aminopurine (2-AP) dinucleotide determined by ultraviolet 2D fluorescence spectroscopy (UV-2D FS).

    PubMed

    Widom, Julia R; Johnson, Neil P; von Hippel, Peter H; Marcus, Andrew H

    2013-02-01

    We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analog of the nucleic acid base adenine. This was accomplished by extending two-dimensional fluorescence spectroscopy (2D FS) - a fluorescence-detected variation of 2D electronic spectroscopy - to excite molecular transitions in the ultraviolet (UV) regime. A collinear sequence of four ultrafast laser pulses centered at 323 nm was used to resonantly excite the coupled transitions of 2-AP dinucleotide. The phases of the optical pulses were continuously swept at kilohertz frequencies, and the ensuing nonlinear fluorescence was phase-synchronously detected at 370 nm. Upon optimization of a point-dipole coupling model to our data, we found that in aqueous buffer the 2-AP dinucleotide adopts an average conformation in which the purine bases are non-helically stacked (center-to-center distance R12 = 3.5 Å ± 0.5 Å, twist angle θ12 = 5° ± 5°), which differs from the conformation of such adjacent bases in duplex DNA. These experiments establish UV-2D FS as a method for examining the local conformations of an adjacent pair of fluorescent nucleotides substituted into specific DNA or RNA constructs, which will serve as a powerful probe to interpret, in structural terms, biologically significant local conformational changes within the nucleic acid framework of protein-nucleic acid complexes.

  4. 2D fluorescence spectroscopy for monitoring ion-exchange membrane based technologies - Reverse electrodialysis (RED).

    PubMed

    Pawlowski, Sylwin; Galinha, Claudia F; Crespo, João G; Velizarov, Svetlozar

    2016-01-01

    Reverse electrodialysis (RED) is one of the emerging, membrane-based technologies for harvesting salinity gradient energy. In RED process, fouling is an undesirable operation constraint since it leads to a decrease of the obtainable net power density due to increasing stack electric resistance and pressure drop. Therefore, early fouling detection is one of the main challenges for successful RED technology implementation. In the present study, two-dimensional (2D) fluorescence spectroscopy was used, for the first time, as a tool for fouling monitoring in RED. Fluorescence excitation-emission matrices (EEMs) of ion-exchange membrane surfaces and of natural aqueous streams were acquired during one month of a RED stack operation. Fouling evolvement on the ion-exchange membrane surfaces was successfully followed by 2D fluorescence spectroscopy and quantified using principal components analysis (PCA). Additionally, the efficiency of cleaning strategy was assessed by measuring the membrane fluorescence emission intensity before and after cleaning. The anion-exchange membrane (AEM) surface in contact with river water showed to be significantly affected due to fouling by humic compounds, which were found to cross through the membrane from the lower salinity (river water) to higher salinity (sea water) stream. The results obtained show that the combined approach of using 2D fluorescence spectroscopy and PCA has a high potential for studying fouling development and membrane cleaning efficiency in ion exchange membrane processes.

  5. Temperature-dependent conformations of a membrane supported zinc porphyrin tweezer by 2D fluorescence spectroscopy.

    PubMed

    Widom, Julia R; Lee, Wonbae; Perdomo-Ortiz, Alejandro; Rappoport, Dmitrij; Molinski, Tadeusz F; Aspuru-Guzik, Alán; Marcus, Andrew H

    2013-07-25

    We studied the equilibrium conformations of a zinc porphyrin tweezer composed of two carboxylphenyl-functionalized zinc tetraphenyl porphyrin subunits connected by a 1,4-butyndiol spacer, which was suspended inside the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) liposomes. By combining phase-modulation two-dimensional fluorescence spectroscopy (2D FS) with linear absorbance and fluorimetry, we determined that the zinc porphyrin tweezer adopts a mixture of folded and extended conformations in the membrane. By fitting an exciton-coupling model to a series of data sets recorded over a range of temperatures (17-85 °C) and at different laser center wavelengths, we determined that the folded form of the tweezer is stabilized by a favorable change in the entropy of the local membrane environment. Our results provide insights toward understanding the balance of thermodynamic factors that govern molecular assembly in membranes.

  6. Parallel online multi-wavelength (2D) fluorescence spectroscopy in each well of a continuously shaken microtiter plate.

    PubMed

    Ladner, Tobias; Beckers, Mario; Hitzmann, Bernd; Büchs, Jochen

    2016-12-01

    Small-scale high-throughput screening devices are becoming increasingly important in bioprocess development. Conventional dipping probes for process monitoring are often too large to be used in these devices. Thus, optical measurements are often the method of choice. Even some parameters that cannot directly be measured by fluorescence become accessible via sensitive fluorescence dyes. However, not all compounds of interest are measurable by this technique. Recent studies applying multi-wavelength (2D) fluorescence spectroscopy in combination with chemometrics have shown that information on numerous analytes is obscured by the fluorescence data. Hitherto, this measurement technique has only been available on the scale of stirred tank fermenters. This work introduces a new device for multi-wavelength (2D) fluorescence spectroscopy in each well of a continuously shaken microtiter plate. Using a combination of spectrograph and CCD detector, the required time per measurement cycle in a 48-well microtiter plate was 0.5 h. Cultures of Hansenula polymorpha and Escherichia coli are monitored. The concentrations of glycerol, glucose and acetate as well as pH are determined using partial least square (PLS) models. Because a pH-sensitive fluorescence dye was not required, no dependency of the pKa of a fluorescence dye exists, and measurements in the low pH range can be obtained.

  7. Conformation and electronic population transfer in membrane-supported self-assembled porphyrin dimers by 2D fluorescence spectroscopy.

    PubMed

    Perdomo-Ortiz, Alejandro; Widom, Julia R; Lott, Geoffrey A; Aspuru-Guzik, Alán; Marcus, Andrew H

    2012-09-06

    Two-dimensional fluorescence spectroscopy (2D FS) is applied to determine the conformation and femtosecond electronic population transfer in a dimer of magnesium meso tetraphenylporphyrin. The dimers are prepared by self-assembly of the monomer within the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine liposomes. A theoretical framework to describe 2D FS experiments is presented, and a direct comparison is made between the observables of this measurement and those of 2D electronic spectroscopy (2D ES). The sensitivity of the method to varying dimer conformation is explored. A global multivariable fitting analysis of linear and 2D FS data indicates that the dimer adopts a "bent T-shaped" conformation. Moreover, the manifold of singly excited excitons undergoes rapid electronic dephasing and downhill population transfer on the time scale of ∼95 fs. The open conformation of the dimer suggests that its self-assembly is favored by an increase in entropy of the local membrane environment.

  8. Electron momentum distribution and singlet-singlet annihilation in the organic anthracene molecular crystals using positron 2D-ACAR and fluorescence spectroscopy.

    PubMed

    Selvakumar, Sellaiyan; Sivaji, Krishnan; Arulchakkaravarthi, Arjunan; Sankar, Sambasivam

    2014-08-14

    We present the mapping of electron momentum distribution (EMD) in a single crystal of anthracene by two-dimensional angular correlation of positron annihilation radiation (2D-ACAR). The projected EMD is explained on the basis of the crystallographic features of the material. The EMD spectra provide information about the positron states and their behavior and also about the hindrance of the positronium (Ps) formation in this material. The EMD has exhibited evidence for the absence of free volume defects. The characteristic EMD features regarding the delocalized electronic states are explained. Further, scintillation characteristics such as fluorescence and time-correlated single photon counting have also been studied. The emission peaks are attributed to vibrational bands of fluorescence emission from the singlet excitons and lifetime components are observed to be due to singlet fission and the singlet-singlet excitons annihilation.

  9. Quantum coherence selective 2D Raman–2D electronic spectroscopy

    PubMed Central

    Spencer, Austin P.; Hutson, William O.; Harel, Elad

    2017-01-01

    Electronic and vibrational correlations report on the dynamics and structure of molecular species, yet revealing these correlations experimentally has proved extremely challenging. Here, we demonstrate a method that probes correlations between states within the vibrational and electronic manifold with quantum coherence selectivity. Specifically, we measure a fully coherent four-dimensional spectrum which simultaneously encodes vibrational–vibrational, electronic–vibrational and electronic–electronic interactions. By combining near-impulsive resonant and non-resonant excitation, the desired fifth-order signal of a complex organic molecule in solution is measured free of unwanted lower-order contamination. A critical feature of this method is electronic and vibrational frequency resolution, enabling isolation and assignment of individual quantum coherence pathways. The vibronic structure of the system is then revealed within an otherwise broad and featureless 2D electronic spectrum. This method is suited for studying elusive quantum effects in which electronic transitions strongly couple to phonons and vibrations, such as energy transfer in photosynthetic pigment–protein complexes. PMID:28281541

  10. Quantum coherence selective 2D Raman-2D electronic spectroscopy

    NASA Astrophysics Data System (ADS)

    Spencer, Austin P.; Hutson, William O.; Harel, Elad

    2017-03-01

    Electronic and vibrational correlations report on the dynamics and structure of molecular species, yet revealing these correlations experimentally has proved extremely challenging. Here, we demonstrate a method that probes correlations between states within the vibrational and electronic manifold with quantum coherence selectivity. Specifically, we measure a fully coherent four-dimensional spectrum which simultaneously encodes vibrational-vibrational, electronic-vibrational and electronic-electronic interactions. By combining near-impulsive resonant and non-resonant excitation, the desired fifth-order signal of a complex organic molecule in solution is measured free of unwanted lower-order contamination. A critical feature of this method is electronic and vibrational frequency resolution, enabling isolation and assignment of individual quantum coherence pathways. The vibronic structure of the system is then revealed within an otherwise broad and featureless 2D electronic spectrum. This method is suited for studying elusive quantum effects in which electronic transitions strongly couple to phonons and vibrations, such as energy transfer in photosynthetic pigment-protein complexes.

  11. Quantum coherence selective 2D Raman-2D electronic spectroscopy.

    PubMed

    Spencer, Austin P; Hutson, William O; Harel, Elad

    2017-03-10

    Electronic and vibrational correlations report on the dynamics and structure of molecular species, yet revealing these correlations experimentally has proved extremely challenging. Here, we demonstrate a method that probes correlations between states within the vibrational and electronic manifold with quantum coherence selectivity. Specifically, we measure a fully coherent four-dimensional spectrum which simultaneously encodes vibrational-vibrational, electronic-vibrational and electronic-electronic interactions. By combining near-impulsive resonant and non-resonant excitation, the desired fifth-order signal of a complex organic molecule in solution is measured free of unwanted lower-order contamination. A critical feature of this method is electronic and vibrational frequency resolution, enabling isolation and assignment of individual quantum coherence pathways. The vibronic structure of the system is then revealed within an otherwise broad and featureless 2D electronic spectrum. This method is suited for studying elusive quantum effects in which electronic transitions strongly couple to phonons and vibrations, such as energy transfer in photosynthetic pigment-protein complexes.

  12. Assessing 2D electrophoretic mobility spectroscopy (2D MOSY) for analytical applications.

    PubMed

    Fang, Yuan; Yushmanov, Pavel V; Furó, István

    2016-12-08

    Electrophoretic displacement of charged entity phase modulates the spectrum acquired in electrophoretic NMR experiments, and this modulation can be presented via 2D FT as 2D mobility spectroscopy (MOSY) spectra. We compare in various mixed solutions the chemical selectivity provided by 2D MOSY spectra with that provided by 2D diffusion-ordered spectroscopy (DOSY) spectra and demonstrate, under the conditions explored, a superior performance of the former method. 2D MOSY compares also favourably with closely related LC-NMR methods. The shape of 2D MOSY spectra in complex mixtures is strongly modulated by the pH of the sample, a feature that has potential for areas such as in drug discovery and metabolomics. Copyright © 2016 The Authors. Magnetic Resonance in Chemistry published by John Wiley & Sons Ltd. StartCopTextCopyright © 2016 The Authors. Magnetic Resonance in Chemistry published by John Wiley & Sons Ltd.

  13. Binding of 7-methoxy-4-(aminomethyl)-coumarin to wild-type and W128F mutant cytochrome P450 2D6 studied by time-resolved fluorescence spectroscopy

    PubMed Central

    Stortelder, Aike; Keizers, Peter H. J.; Oostenbrink, Chris; De Graaf, Chris; De Kruijf, Petra; Vermeulen, Nico P. E.; Gooijer, Cees; Commandeur, Jan N. M.; Van Der Zwan, Gert

    2005-01-01

    Enzyme structure and dynamics may play a main role in substrate binding and the subsequent steps in the CYP (cytochrome P450) catalytic cycle. In the present study, changes in the structure of human CYP2D6 upon binding of the substrate are studied using steady-state and time-resolved fluorescence methods, focusing not only on the emission of the tryptophan residues, but also on emission of the substrate. As a substrate, MAMC [7-methoxy-4-(aminomethyl)-coumarin] was selected, a compound exhibiting native fluorescence. As well as the wild-type, the W128F (Trp128→Phe) mutant of CYP2D6 was studied. After binding, a variety of energy transfer possibilities exist, and molecular dynamics simulations were performed to calculate distances and relative orientations of donors and acceptors. Energy transfer from Trp128 to haem appeared to be important; its emission was related to the shortest of the three average tryptophan fluorescence lifetimes observed for CYP2D6. MAMC to haem energy transfer was very efficient as well: when bound in the active site, the emission of MAMC was fully quenched. Steady-state anisotropy revealed that besides the MAMC in the active site, another 2.4% of MAMC was bound outside of the active site to wild-type CYP2D6. The tryptophan residues in CYP2D6 appeared to be less accessible for the external quenchers iodide and acrylamide in presence of MAMC, indicating a tightening of the enzyme structure upon substrate binding. However, the changes in the overall enzyme structure were not very large, since the emission characteristics of the enzyme were not very different in the presence of MAMC. PMID:16190863

  14. Optimizing sparse sampling for 2D electronic spectroscopy

    NASA Astrophysics Data System (ADS)

    Roeding, Sebastian; Klimovich, Nikita; Brixner, Tobias

    2017-02-01

    We present a new data acquisition concept using optimized non-uniform sampling and compressed sensing reconstruction in order to substantially decrease the acquisition times in action-based multidimensional electronic spectroscopy. For this we acquire a regularly sampled reference data set at a fixed population time and use a genetic algorithm to optimize a reduced non-uniform sampling pattern. We then apply the optimal sampling for data acquisition at all other population times. Furthermore, we show how to transform two-dimensional (2D) spectra into a joint 4D time-frequency von Neumann representation. This leads to increased sparsity compared to the Fourier domain and to improved reconstruction. We demonstrate this approach by recovering transient dynamics in the 2D spectrum of a cresyl violet sample using just 25% of the originally sampled data points.

  15. Broadband THz Spectroscopy of 2D Nanoscale Materials

    NASA Astrophysics Data System (ADS)

    Chen, Lu; Tripathi, Shivendra; Huang, Mengchen; Hsu, Jen-Feng; D'Urso, Brian; Lee, Hyungwoo; Eom, Chang-Beom; Irvin, Patrick; Levy, Jeremy

    Two-dimensional (2D) materials such as graphene and transition-metal dichalcogenides (TMDC) have attracted intense research interest in the past decade. Their unique electronic and optical properties offer the promise of novel optoelectronic applications in the terahertz regime. Recently, generation and detection of broadband terahertz (10 THz bandwidth) emission from 10-nm-scale LaAlO3/SrTiO3 nanostructures created by conductive atomic force microscope (c-AFM) lithography has been demonstrated . This unprecedented control of THz emission at 10 nm length scales creates a pathway toward hybrid THz functionality in 2D-material/LaAlO3/SrTiO3 heterostructures. Here we report initial efforts in THz spectroscopy of 2D nanoscale materials with resolution comparable to the dimensions of the nanowire (10 nm). Systems under investigation include graphene, single-layer molybdenum disulfide (MoS2), and tungsten diselenide (WSe2) nanoflakes. 1. Y. Ma, et al., Nano Lett. 13, 2884 (2013). We gratefully acknowledge financial support from the following agencies and grants: AFOSR (FA9550-12-1-0268 (JL, PRI), FA9550-12-1-0342 (CBE)), ONR (N00014-13-1-0806 (JL, CBE), N00014-15-1-2847 (JL)), NSF DMR-1124131 (JL, CBE) and DMR-1234096 (CBE).

  16. Smartphone fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Yu, Hojoeng; Tan, Yafang; Cunningham, Brian T.

    2014-03-01

    We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of a specific nucleic acid sequences in a liquid test sample. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route towards portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

  17. Peak width issues with generalised 2D correlation NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Kirwan, Gemma M.; Adams, Michael J.

    2008-12-01

    Two-dimensional spectral correlation analysis is shown to be sensitive to fluctuations in spectral peak width as a function of perturbation variable. This is particularly significant where peak width fluctuations are of similar order of magnitude as the peak width values themselves and where changes in peak width are not random but are, for example, proportional to intensity. In such cases these trends appear in the asynchronous matrix as false peaks that serve to interfere with interpretation of the data. Complex, narrow band spectra such as provided by 1H NMR spectroscopy are demonstrated to be prone to such interference. 2D correlation analysis was applied to a series of NMR spectra corresponding to a commercial wine fermentation, in which the samples collected over a period of several days exhibit dramatic changes in concentration of minor and major components. The interference due to changing peak width effects is eliminated by synthesizing the recorded spectra using a constant peak width value prior to performing 2D correlation analysis.

  18. Coherent 2D Spectroscopy and Control of Molecular Complexes

    NASA Astrophysics Data System (ADS)

    Brixner, Tobias

    2007-03-01

    Coherent two-dimensional femtosecond spectroscopy is used to investigate electronic couplings within molecular complexes. Third-order optical response functions are measured in a non-collinear three-pulse photon echo geometry with heterodyne signal detection. In combination with suitable simulations this allows recovering the delocalization of excited-state wavefunctions, their coupling, and the corresponding energy transport pathways, with nanometer spatial and femtosecond temporal resolution. Examples of multichromophoric systems are the FMO and the LH3 light-harvesting complexes from green sulfur bacteria and purple bacteria, respectively, for which energy transfer processes have been determined. Additional challenges arise if one is interested in the spectroscopy of photochemical rather than photophysical processes in molecular complexes: The product yields attained by a single femtosecond laser pulse are often very small, and hence time-dependent signals are hard to measure with good signal-to-noise ratio. In the context of coherent control, this implies that bond-breaking photochemistry in liquids is still difficult despite the many successes of optimal control in gas-phase photodissociation. In a novel accumulative scheme, macroscopic amounts of stable photoproducts are generated in an optimal fashion and with high product detection sensitivity. In connection with time-resolved spectroscopy, the accumulative scheme furthermore provides kinetic information on the pathways of low-efficiency chemical reaction channels. This was applied to investigate the photoconversion of green fluorescent protein.

  19. 2-D fluorescence lifetime imaging using a time-gated image intensifier

    NASA Astrophysics Data System (ADS)

    Dowling, K.; Hyde, S. C. W.; Dainty, J. C.; French, P. M. W.; Hares, J. D.

    1997-02-01

    We report a 2-D fluorescence lifetime imaging system based on a time-gated image intensifier and a Cr:LiSAF regenerative amplifier. We have demonstrated 185 ps temporal resolution. The deleterious effects of optical scattering are demonstrated.

  20. Double resonance rotational spectroscopy of CH2D+

    NASA Astrophysics Data System (ADS)

    Töpfer, Matthias; Jusko, Pavol; Schlemmer, Stephan; Asvany, Oskar

    2016-09-01

    Context. Deuterated forms of CH are thought to be responsible for deuterium enrichment in lukewarm astronomical environments. There is no unambiguous detection of CH2D+ in space to date. Aims: Four submillimetre rotational lines of CH2D+ are documented in the literature. Our aim is to present a complete dataset of highly resolved rotational lines, including millimetre (mm) lines needed for a potential detection. Methods: We used a low-temperature ion trap and applied a novel IR-mm-wave double resonance method to measure the rotational lines of CH2D+. Results: We measured 21 low-lying (J ≤ 4) rotational transitions of CH2D+ between 23 GHz and 1.1 THz with accuracies close to 2 ppb.

  1. Monitoring guanidinium-induced structural changes in ribonuclease proteins using Raman spectroscopy and 2D correlation analysis.

    PubMed

    Brewster, Victoria L; Ashton, Lorna; Goodacre, Royston

    2013-04-02

    Assessing the stability of proteins by comparing their unfolding profiles is a very important characterization and quality control step for any biopharmaceutical, and this is usually measured by fluorescence spectroscopy. In this paper we propose Raman spectroscopy as a rapid, noninvasive alternative analytical method and we shall show this has enhanced sensitivity and can therefore reveal very subtle protein conformational changes that are not observed with fluorescence measurements. Raman spectroscopy is a powerful nondestructive method that has a strong history of applications in protein characterization. In this work we describe how Raman microscopy can be used as a fast and reliable method of tracking protein unfolding in the presence of a chemical denaturant. We have compared Raman spectroscopic data to the equivalent samples analyzed using fluorescence spectroscopy in order to validate the Raman approach. Calculations from both Raman and fluorescence unfolding curves of [D]50 values and Gibbs free energy correlate well with each other and more importantly agree with the values found in the literature for these proteins. In addition, 2D correlation analysis has been performed on both Raman and fluorescence data sets in order to allow further comparisons of the unfolding behavior indicated by each method. As many biopharmaceuticals are glycosylated in order to be functional, we compare the unfolding profiles of a protein (RNase A) and a glycoprotein (RNase B) as measured by Raman spectroscopy and discuss the implications that glycosylation has on the stability of the protein.

  2. Human erythrocytes analyzed by generalized 2D Raman correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Wesełucha-Birczyńska, Aleksandra; Kozicki, Mateusz; Czepiel, Jacek; Łabanowska, Maria; Nowak, Piotr; Kowalczyk, Grzegorz; Kurdziel, Magdalena; Birczyńska, Malwina; Biesiada, Grażyna; Mach, Tomasz; Garlicki, Aleksander

    2014-07-01

    The most numerous elements of the blood cells, erythrocytes, consist mainly of two components: homogeneous interior filled with hemoglobin and closure which is the cell membrane. To gain insight into their specific properties we studied the process of disintegration, considering these two constituents, and comparing the natural aging process of human healthy blood cells. MicroRaman spectra of hemoglobin within the single RBC were recorded using 514.5, and 785 nm laser lines. The generalized 2D correlation method was applied to analyze the collected spectra. The time passed from blood donation was regarded as an external perturbation. The time was no more than 40 days according to the current storage limit of blood banks, although, the average RBC life span is 120 days. An analysis of the prominent synchronous and asynchronous cross peaks allow us to get insight into the mechanism of hemoglobin decomposition. Appearing asynchronous cross-peaks point towards globin and heme separation from each other, while synchronous shows already broken globin into individual amino acids. Raman scattering analysis of hemoglobin "wrapping", i.e. healthy erythrocyte ghosts, allows for the following peculiarity of their behavior. The increasing power of the excitation laser induced alterations in the assemblage of membrane lipids. 2D correlation maps, obtained with increasing laser power recognized as an external perturbation, allows for the consideration of alterations in the erythrocyte membrane structure and composition, which occurs first in the proteins. Cross-peaks were observed indicating an asynchronous correlation between the senescent-cell antigen (SCA) and heme or proteins vibrations. The EPR spectra of the whole blood was analyzed regarding time as an external stimulus. The 2D correlation spectra points towards participation of the selected metal ion centers in the disintegration process.

  3. 2D map projections for visualization and quantitative analysis of 3D fluorescence micrographs

    PubMed Central

    Sendra, G. Hernán; Hoerth, Christian H.; Wunder, Christian; Lorenz, Holger

    2015-01-01

    We introduce Map3-2D, a freely available software to accurately project up to five-dimensional (5D) fluorescence microscopy image data onto full-content 2D maps. Similar to the Earth’s projection onto cartographic maps, Map3-2D unfolds surface information from a stack of images onto a single, structurally connected map. We demonstrate its applicability for visualization and quantitative analyses of spherical and uneven surfaces in fixed and dynamic live samples by using mammalian and yeast cells, and giant unilamellar vesicles. Map3-2D software is available at http://www.zmbh.uni-heidelberg.de//Central_Services/Imaging_Facility/Map3-2D.html. PMID:26208256

  4. Laser irradiated fluorescent perfluorocarbon microparticles in 2-D and 3-D breast cancer cell models

    NASA Astrophysics Data System (ADS)

    Niu, Chengcheng; Wang, Long; Wang, Zhigang; Xu, Yan; Hu, Yihe; Peng, Qinghai

    2017-03-01

    Perfluorocarbon (PFC) droplets were studied as new generation ultrasound contrast agents via acoustic or optical droplet vaporization (ADV or ODV). Little is known about the ODV irradiated vaporization mechanisms of PFC-microparticle complexs and the stability of the new bubbles produced. In this study, fluorescent perfluorohexane (PFH) poly(lactic-co-glycolic acid) (PLGA) particles were used as a model to study the process of particle vaporization and bubble stability following excitation in two-dimensional (2-D) and three-dimensional (3-D) cell models. We observed localization of the fluorescent agent on the microparticle coating material initially and after vaporization under fluorescence microscopy. Furthermore, the stability and growth dynamics of the newly created bubbles were observed for 11 min following vaporization. The particles were co-cultured with 2-D cells to form 3-D spheroids and could be vaporized even when encapsulated within the spheroids via laser irradiation, which provides an effective basis for further work.

  5. Fluorescence spectroscopy applied to orange trees

    NASA Astrophysics Data System (ADS)

    Marcassa, L. G.; Gasparoto, M. C. G.; Belasque, J., Jr.; Lins, E. C.; Dias Nunes, F.; Bagnato, V. S.

    2006-05-01

    In this work, we have applied laser-induced fluorescence spectroscopy to investigate biological processes in orange trees (Citrus aurantium L.). We have chosen to investigate water stress and Citrus Canker, which is a disease caused by the Xanthomonas axonopodis pv. citri bacteria. The fluorescence spectroscopy was investigated by using as an excitation source a 442-nm 15-mW HeCd gas multimode discharge laser and a 532-nm 10-mW Nd3+:YAG laser. The stress manifestation was detected by the variation of fluorescence ratios of the leaves at different wavelengths. The fluorescence ratios present a significant variation, showing the possibility to observe water stress by fluorescence spectrum. The Citrus Canker’s contaminated leaves were discriminated from the healthy leaves using a more complex analysis of the fluorescence spectra. However, we were unable to discriminate it from another disease, and new fluorescence experiments are planned for the future.

  6. Multiphoton, optical fiber-based fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Bereś-Pawlik, ElŻbieta; Stawska, Hanna; Popenda, Maciej; Pajewski, Łukasz; Malinowska, Natalia; Hossa, Robert

    2016-12-01

    This paper presents investigation of normal and cancerous tissue by the means of one and two photon fluorescence spectroscopy. A comparison those methods has been conducted, allowing for eventual determination of granting the best possible diagnostic results.

  7. Targeted fluorescence imaging enhanced by 2D materials: a comparison between 2D MoS2 and graphene oxide.

    PubMed

    Xie, Donghao; Ji, Ding-Kun; Zhang, Yue; Cao, Jun; Zheng, Hu; Liu, Lin; Zang, Yi; Li, Jia; Chen, Guo-Rong; James, Tony D; He, Xiao-Peng

    2016-08-04

    Here we demonstrate that 2D MoS2 can enhance the receptor-targeting and imaging ability of a fluorophore-labelled ligand. The 2D MoS2 has an enhanced working concentration range when compared with graphene oxide, resulting in the improved imaging of both cell and tissue samples.

  8. Fast, accurate 2D-MR relaxation exchange spectroscopy (REXSY): Beyond compressed sensing

    NASA Astrophysics Data System (ADS)

    Bai, Ruiliang; Benjamini, Dan; Cheng, Jian; Basser, Peter J.

    2016-10-01

    Previously, we showed that compressive or compressed sensing (CS) can be used to reduce significantly the data required to obtain 2D-NMR relaxation and diffusion spectra when they are sparse or well localized. In some cases, an order of magnitude fewer uniformly sampled data were required to reconstruct 2D-MR spectra of comparable quality. Nonetheless, this acceleration may still not be sufficient to make 2D-MR spectroscopy practicable for many important applications, such as studying time-varying exchange processes in swelling gels or drying paints, in living tissue in response to various biological or biochemical challenges, and particularly for in vivo MRI applications. A recently introduced framework, marginal distributions constrained optimization (MADCO), tremendously accelerates such 2D acquisitions by using a priori obtained 1D marginal distribution as powerful constraints when 2D spectra are reconstructed. Here we exploit one important intrinsic property of the 2D-MR relaxation exchange spectra: the fact that the 1D marginal distributions of each 2D-MR relaxation exchange spectrum in both dimensions are equal and can be rapidly estimated from a single Carr-Purcell-Meiboom-Gill (CPMG) or inversion recovery prepared CPMG measurement. We extend the MADCO framework by further proposing to use the 1D marginal distributions to inform the subsequent 2D data-sampling scheme, concentrating measurements where spectral peaks are present and reducing them where they are not. In this way we achieve compression or acceleration that is an order of magnitude greater than that in our previous CS method while providing data in reconstructed 2D-MR spectral maps of comparable quality, demonstrated using several simulated and real 2D T2 - T2 experimental data. This method, which can be called "informed compressed sensing," is extendable to other 2D- and even ND-MR exchange spectroscopy.

  9. Radiofrequency Spectroscopy and Thermodynamics of Fermi Gases in the 2D to Quasi-2D Dimensional Crossover

    NASA Astrophysics Data System (ADS)

    Cheng, Chingyun; Kangara, Jayampathi; Arakelyan, Ilya; Thomas, John

    2016-05-01

    We tune the dimensionality of a strongly interacting degenerate 6 Li Fermi gas from 2D to quasi-2D, by adjusting the radial confinement of pancake-shaped clouds to control the radial chemical potential. In the 2D regime with weak radial confinement, the measured pair binding energies are in agreement with 2D-BCS mean field theory, which predicts dimer pairing energies in the many-body regime. In the qausi-2D regime obtained with increased radial confinement, the measured pairing energy deviates significantly from 2D-BCS theory. In contrast to the pairing energy, the measured radii of the cloud profiles are not fit by 2D-BCS theory in either the 2D or quasi-2D regimes, but are fit in both regimes by a beyond mean field polaron-model of the free energy. Supported by DOE, ARO, NSF, and AFOSR.

  10. Low-frequency phased-array 2D fluorescence localization in breast cancer detection

    NASA Astrophysics Data System (ADS)

    Liu, Qian; Chen, Yu; Chance, Britton; Luo, Qingming

    2003-12-01

    A method for rapid, non-invasive 2D fluorescence localization of breast cancer using low frequency phased array near-infrared technique is presented in this article. In our study, we have developed a dual-channel fluorescence detection system to locate breast cancer. This system consists two pair of in-phase and out-of-phase light emitting diodes (LEDs) as the light sources and Photomultiplier Tube (PMT) as the detector. Two null planes generated by cancellation of diffusion photon density waves (DPDW) will indicate the 2D position of breast cancer with exogenous contrast agents. The fluorescent contrast agent used in this study is Indocyanine Green (ICG) and the minimum amount of ICG detected by our system is 0.5 μM. With the 2 cm separation of sources and detector, the maximum depth our system can detect is 10 mm. The whole system is in compact size and portable. Phantom experiments show that the system can provide real time detection and localization of small hidden absorbing-fluorescent objects inside the highly scattering medium with high accuracy of +/-3 mm. The potential application is that it is low-cost and can be used for breast cancer localization as operation aid and self-examination.

  11. Fluorescence fluctuation spectroscopy in reduced detection volumes.

    PubMed

    Blom, H; Kastrup, L; Eggeling, C

    2006-02-01

    Fluorescence fluctuation spectroscopy is a versatile technique applied to in vitro and in vivo investigations of biochemical processes such as interactions, mobilities or densities with high specifity and sensitivity. The prerequisite of this dynamical fluorescence technique is to have, at a time, only few fluorescent molecules in the detection volume in order to generate significant fluorescence fluctuations. For usual confocal fluorescence microscopy this amounts to a useful concentration in the nanomolar range. The concentration of many biomolecules in living cell or on cell membranes is, however, often quite high, usually in the micro- to the millimolar range. To allow fluctuation spectroscopy and track intracellular interaction or localization of single fluorescently labeled biomolecules in such crowded environments, development of detection volumes with nanoscale resolution is necessary. As diffraction prevents this in the case of light microscopy, new (non-invasive) optical concepts have been developed. In this mini-review article we present recent advancements, implemented to decrease the detection volume below that of normal fluorescence microscopy. Especially, their combination with fluorescence fluctuation spectroscopy is emphasized.

  12. Fluorescence spectroscopy of rhodopsins: Insights and approaches

    PubMed Central

    Alexiev, Ulrike; Farrens, David L.

    2014-01-01

    Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy. PMID:24183695

  13. Comparing the heterogeneity of copper-binding characteristics for two different-sized soil humic acid fractions using fluorescence quenching combined with 2D-COS.

    PubMed

    Hur, Jin; Lee, Bo-Mi

    2011-01-01

    Heterogeneous distributions of copper-binding characteristics were compared for two ultrafiltered size fractions of a soil HA using fluorescence quenching combined with two-dimensional correlation spectroscopy (2D-COS). The apparent shapes of the original synchronous fluorescence spectra and the extent of the fluorescence quenching upon the addition of copper were similar for the two fractions. The stability constants calculated at their highest peaks were not significantly different. However, the 2D-COS results revealed that the fluorescence quenching behaviors were strongly affected by the associated wavelengths and the fraction's size. The spectral change preferentially occurred in the wavelength order of 467 nm → 451 nm → 357 nm for the 1-10 K fraction and of 376 nm → 464 nm for the >100 K fraction. The extent of the binding affinities exactly followed the sequential orders interpreted from the 2D-COS, and they exhibited the distinctive ranges of the logarithmic values from 5.86 to 4.91 and from 6.48 to 5.95 for the 1-10 K and the >100 K fractions, respectively. Our studies demonstrated that fluorescence quenching combined with 2D-COS could be successfully utilized to give insight into the chemical heterogeneity associated with metal-binding sites within the relatively homogeneous HA size fractions.

  14. Laser irradiated fluorescent perfluorocarbon microparticles in 2-D and 3-D breast cancer cell models

    PubMed Central

    Niu, Chengcheng; Wang, Long; Wang, Zhigang; Xu, Yan; Hu, Yihe; Peng, Qinghai

    2017-01-01

    Perfluorocarbon (PFC) droplets were studied as new generation ultrasound contrast agents via acoustic or optical droplet vaporization (ADV or ODV). Little is known about the ODV irradiated vaporization mechanisms of PFC-microparticle complexs and the stability of the new bubbles produced. In this study, fluorescent perfluorohexane (PFH) poly(lactic-co-glycolic acid) (PLGA) particles were used as a model to study the process of particle vaporization and bubble stability following excitation in two-dimensional (2-D) and three-dimensional (3-D) cell models. We observed localization of the fluorescent agent on the microparticle coating material initially and after vaporization under fluorescence microscopy. Furthermore, the stability and growth dynamics of the newly created bubbles were observed for 11 min following vaporization. The particles were co-cultured with 2-D cells to form 3-D spheroids and could be vaporized even when encapsulated within the spheroids via laser irradiation, which provides an effective basis for further work. PMID:28262671

  15. Interferometric 2D Sum Frequency Generation Spectroscopy Reveals Structural Heterogeneity of Catalytic Monolayers on Transparent Materials.

    PubMed

    Vanselous, Heather; Stingel, Ashley M; Petersen, Poul B

    2017-02-16

    Molecular monolayers exhibit structural and dynamical properties that are different from their bulk counterparts due to their interaction with the substrate. Extracting these distinct properties is crucial for a better understanding of processes such as heterogeneous catalysis and interfacial charge transfer. Ultrafast nonlinear spectroscopic techniques such as 2D infrared (2D IR) spectroscopy are powerful tools for understanding molecular dynamics in complex bulk systems. Here, we build on technical advancements in 2D IR and heterodyne-detected sum frequency generation (SFG) spectroscopy to study a CO2 reduction catalyst on nanostructured TiO2 with interferometric 2D SFG spectroscopy. Our method combines phase-stable heterodyne detection employing an external local oscillator with a broad-band pump pulse pair to provide the first high spectral and temporal resolution 2D SFG spectra of a transparent material. We determine the overall molecular orientation of the catalyst and find that there is a static structural heterogeneity reflective of different local environments at the surface.

  16. Two-dimensional fluorescence spectroscopy for application in biotechnology

    NASA Astrophysics Data System (ADS)

    Lindemann, Carsten; Marose, S.; Scheper, Thomas-Helmut; Nielsen, Hans O.; Hitzmann, Bernd; Belgardt, K.-H.

    1999-02-01

    A wide range of excitation and emission wavelengths is measured using the technique of two-dimensional (2D-) fluorescence spectroscopy. In a single, so called, two- dimensional fluorescence spectrum several biogenic fluorophors like proteins, vitamins and coenzymes can be detected simultaneously. This can give important information for bioprocess monitoring and control. An optical sensor (BioViewR) for on line fluorescence measurements at industrial (bio)-processes was used to get the results presented in this paper. This BioViewR-sensor is optimized to work in the harsh environment of production sites in biotechnological industry and -- using an optical light guide system with open-end detection -- it is very well suited for in vivo measurements, because it is non-invasive and the on line data can be performed in-situ.

  17. Micro-reflectance and transmittance spectroscopy: a versatile and powerful tool to characterize 2D materials

    NASA Astrophysics Data System (ADS)

    Frisenda, Riccardo; Niu, Yue; Gant, Patricia; Molina-Mendoza, Aday J.; Schmidt, Robert; Bratschitsch, Rudolf; Liu, Jinxin; Fu, Lei; Dumcenco, Dumitru; Kis, Andras; Perez De Lara, David; Castellanos-Gomez, Andres

    2017-02-01

    Optical spectroscopy techniques such as differential reflectance and transmittance have proven to be very powerful techniques for studying 2D materials. However, a thorough description of the experimental setups needed to carry out these measurements is lacking in the literature. We describe a versatile optical microscope setup for carrying out differential reflectance and transmittance spectroscopy in 2D materials with a lateral resolution of ~1 µm in the visible and near-infrared part of the spectrum. We demonstrate the potential of the presented setup to determine the number of layers of 2D materials and characterize their fundamental optical properties, such as excitonic resonances. We illustrate its performance by studying mechanically exfoliated and chemical vapor-deposited transition metal dichalcogenide samples.

  18. Fluorescence spectroscopy for neoplasms control

    NASA Astrophysics Data System (ADS)

    Bratchenko, I. A.; Kristoforova, Yu. A.; Myakinin, O. O.; Artemyev, D. N.; Kozlov, S. V.; Moryatov, A. A.; Zakharov, V. P.

    2016-04-01

    Investigation of malignant skin tumors diagnosis was performed involving two setups for native tissues fluorescence control in visible and near infrared regions. Combined fluorescence analysis for skin malignant melanomas and basal cell carcinomas was performed. Autofluorescence spectra of normal skin and oncological pathologies stimulated by 457 nm and 785 nm lasers were registered for 74 skin tissue samples. Spectra of 10 melanomas and 27 basal cell carcinomas were registered ex vivo. Skin tumors analysis was made on the basis of autofluorescence spectra intensity and curvature for analysis of porphyrins, lipo-pigments, flavins and melanin. Separation of melanomas and basal cell carcinomas was performed on the basis of discriminant analysis. Overall accuracy of basal cell carcinomas and malignant melanomas separation in current study reached 86.5% with 70% sensitivity and 92.6% specificity.

  19. Fluorescence Spectroscopy with Surface Plasmon Excitation

    NASA Astrophysics Data System (ADS)

    Neumann, T.; Kreiter, M.; Knoll, W.

    In recent years, much effort has been directed towards the development of optical biosensors. While direct sensors are capable of monitoring the presence of an analyte without the use of labelling groups, the class of indirect sensors exploits the signal enhancement caused by bound marker molecules. Surface plasmon spectroscopy (SPS) as a direct detection method [1] is known to lack sensitivity for monitoring of low molecular mass analytes. In order to enhance the sensitivity and to improve the detection limit the technique was combined with fluorescence detection schemes in surface plasmon fluorescence spectroscopy (SPFS), as described recently [2]. Here, we briefly review the theory of plasmon excitation and the experimental realization of SPFS.

  20. Online fluorescence suppression in modulated Raman spectroscopy.

    PubMed

    De Luca, Anna Chiara; Mazilu, Michael; Riches, Andrew; Herrington, C Simon; Dholakia, Kishan

    2010-01-15

    Label-free chemical characterization of single cells is an important aim for biomedical research. Standard Raman spectroscopy provides intrinsic biochemical markers for noninvasive analysis of biological samples but is often hindered by the presence of fluorescence background. In this paper, we present an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background. The method is based on the principle that the fluorescence background does not change whereas the Raman scattering is shifted by the periodical modulation of the laser wavelength. Exploiting this physical property and importantly the multichannel lock-in detection of the Raman signal, the modulation technique fulfills the requirements of an effective fluorescence subtraction method. Indeed, once the synchronization and calibration procedure is performed, minimal user intervention is required, making the method online and less time-consuming than the other fluorescent suppression methods. We analyze the modulated Raman signal and shifted excitation Raman difference spectroscopy (SERDS) signal of 2 mum-sized polystyrene beads suspended in a solution of fluorescent dye as a function of modulation rate. We show that the signal-to-noise ratio of the modulated Raman spectra at the highest modulation rate is 3 times higher than the SERDS one. To finally evaluate the real benefits of the modulated Raman spectroscopy, we apply our technique to Chinese hamster ovary cells (CHO). Specifically, by analyzing separate spectra from the membrane, cytoplasm, and nucleus of CHO cells, we demonstrate the ability of this method to obtain localized sensitive chemical information from cells, away from the interfering fluorescence background. In particular, statistical analysis of the Raman data and classification using PCA (principal component analysis) indicate that our method allows us to distinguish between different cell locations with higher sensitivity and

  1. Xanthines Studied via Femtosecond Fluorescence Spectroscopy.

    PubMed

    Changenet-Barret, Pascale; Kovács, Lajos; Markovitsi, Dimitra; Gustavsson, Thomas

    2016-12-03

    Xanthines represent a wide class of compounds closely related to the DNA bases adenine and guanine. Ubiquitous in the human body, they are capable of replacing natural bases in double helices and give rise to four-stranded structures. Although the use of their fluorescence for analytical purposes was proposed, their fluorescence properties have not been properly characterized so far. The present paper reports the first fluorescence study of xanthine solutions relying on femtosecond spectroscopy. Initially, we focus on 3-methylxanthine, showing that this compound exhibits non-exponential fluorescence decays with no significant dependence on the emission wavelength. The fluorescence quantum yield (3 × 10(-4)) and average decay time (0.9 ps) are slightly larger than those found for the DNA bases. Subsequently, we compare the dynamical fluorescence properties of seven mono-, di- and tri-methylated derivatives. Both the fluorescence decays and fluorescence anisotropies vary only weakly with the site and the degree of methylation. These findings are in line with theoretical predictions suggesting the involvement of several conical intersections in the relaxation of the lowest singlet excited state.

  2. Ultraviolet, Visible, and Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Penner, Michael H.

    Spectroscopy in the ultraviolet-visible (UV-Vis) range is one of the most commonly encountered laboratory techniques in food analysis. Diverse examples, such as the quantification of macrocomponents (total carbohydrate by the phenol-sulfuric acid method), quantification of microcomponents, (thiamin by the thiochrome fluorometric procedure), estimates of rancidity (lipid oxidation status by the thiobarbituric acid test), and surveillance testing (enzyme-linked immunoassays), are presented in this text. In each of these cases, the analytical signal for which the assay is based is either the emission or absorption of radiation in the UV-Vis range. This signal may be inherent in the analyte, such as the absorbance of radiation in the visible range by pigments, or a result of a chemical reaction involving the analyte, such as the colorimetric copper-based Lowry method for the analysis of soluble protein.

  3. NMR Analysis of Unknowns: An Introduction to 2D NMR Spectroscopy

    ERIC Educational Resources Information Center

    Alonso, David E.; Warren, Steven E.

    2005-01-01

    A study combined 1D (one-dimensional) and 2D (two-dimensional) NMR spectroscopy to solve structural organic problems of three unknowns, which include 2-, 3-, and 4-heptanone. Results showed [to the first power]H NMR and [to the thirteenth power]C NMR signal assignments for 2- and 3-heptanone were more challenging than for 4-heptanone owing to the…

  4. Differentiating tissue by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Woessner, Stefan; Huen, Julien; Malthan, Dirk

    2004-03-01

    A common problem in several surgical applications is the lack of navigational information. Most often, the only source of information about the location of crucial structures, in relation to the surgical instrument, is the visible and tactile sensory input of the surgeon. In some cases, this leads to time-consuming procedures and a high risk for the patient. Therefore, we developed a spectroscopic sensor system for automatic differentiation between several tissue types. For example in milling processes, a sensor that is able to detect bone in contrast to nerve or vein tissue can be used to control the milling process. We showed exemplarily for the cochlea implant, a typical ENT-surgery, that with the help of our sensor system, the milling of bone can be accelerated without increasing the risk for the patient. It is also possible to use this type of sensor system in the area of medical robotics in soft-tissue applications. With real-time information, a continuous registration can take place, in contrast to a registration that is done using static preoperatively acquired images. We showed that our sensor system can be used to dynamically update the location of the patient in relation to CT or MR-images. In conclusion, we have been able to show that well-known spectroscopy sensors can be used to open new possibilities in medical treatment with and without the use of robotics.

  5. Single-scan 2D NMR: An Emerging Tool in Analytical Spectroscopy

    PubMed Central

    Giraudeau, Patrick; Frydman, Lucio

    2016-01-01

    Two-dimensional Nuclear Magnetic Resonance (2D NMR) spectroscopy is widely used in chemical and biochemical analyses. Multidimensional NMR is also witnessing an increased use in quantitative and metabolic screening applications. Conventional 2D NMR experiments, however, are affected by inherently long acquisition durations, arising from their need to sample the frequencies involved along their indirect domains in an incremented, scan-by-scan nature. A decade ago a so-called “ultrafast” (UF) approach was proposed, capable to deliver arbitrary 2D NMR spectra involving any kind of homo- or hetero-nuclear correlations, in a single scan. During the intervening years the performance of this sub-second 2D NMR methodology has been greatly improved, and UF 2D NMR is rapidly becoming a powerful analytical tool witnessing an expanded scope of applications. The present reviews summarizes the principles and the main developments which have contributed to the success of this approach, and focuses on applications which have been recently demonstrated in various areas of analytical chemistry –from the real time monitoring of chemical and biochemical processes, to extensions in hyphenated techniques and in quantitative applications. PMID:25014342

  6. Fluorescence spectroscopy for wastewater monitoring: A review.

    PubMed

    Carstea, Elfrida M; Bridgeman, John; Baker, Andy; Reynolds, Darren M

    2016-05-15

    Wastewater quality is usually assessed using physical, chemical and microbiological tests, which are not suitable for online monitoring, provide unreliable results, or use hazardous chemicals. Hence, there is an urgent need to find a rapid and effective method for the evaluation of water quality in natural and engineered systems and for providing an early warning of pollution events. Fluorescence spectroscopy has been shown to be a valuable technique to characterize and monitor wastewater in surface waters for tracking sources of pollution, and in treatment works for process control and optimization. This paper reviews the current progress in applying fluorescence to assess wastewater quality. Studies have shown that, in general, wastewater presents higher fluorescence intensity compared to natural waters for the components associated with peak T (living and dead cellular material and their exudates) and peak C (microbially reprocessed organic matter). Furthermore, peak T fluorescence is significantly reduced after the biological treatment process and peak C is almost completely removed after the chlorination and reverse osmosis stages. Thus, simple fluorometers with appropriate wavelength selectivity, particularly for peaks T and C could be used for online monitoring in wastewater treatment works. This review also shows that care should be taken in any attempt to identify wastewater pollution sources due to potential overlapping fluorophores. Correlations between fluorescence intensity and water quality parameters such as biochemical oxygen demand (BOD) and total organic carbon (TOC) have been developed and dilution of samples, typically up to ×10, has been shown to be useful to limit inner filter effect. It has been concluded that the following research gaps need to be filled: lack of studies on the on-line application of fluorescence spectroscopy in wastewater treatment works and lack of data processing tools suitable for rapid correction and extraction of

  7. Review of applications of fluorescence excitation spectroscopy to dermatology.

    PubMed

    Franco, W; Gutierrez-Herrera, E; Kollias, N; Doukas, A

    2016-03-01

    Endogenous molecules that exhibit fluorescence hold the potential to serve as reporters of tissue structure, activity and physiology. Fluorescence excitation spectroscopy is one means to measure and express tissue's innate fluorescence. This review focuses on the application of endogenous fluorescence ultraviolet excitation spectroscopy to dermatology.

  8. Dual-focus fluorescence correlation spectroscopy.

    PubMed

    Pieper, Christoph; Weiß, Kerstin; Gregor, Ingo; Enderlein, Jörg

    2013-01-01

    This chapter introduces into the technique of dual-focus fluorescence correlation spectroscopy or 2fFCS. In 2fFCS, the fluorescence signals generated in two laterally shifted but overlapping focal regions are auto- and crosscorrelated. The resulting correlation curves are then used to determine diffusion coefficients of fluorescent molecules or particles in solutions or membranes. Moreover, the technique can also be used for noninvasively measuring flow-velocity profiles in three dimensions. Because the distance between the focal regions is precisely known and not changed by most optical aberrations, this provides an accurate and immutable external length scale for determining diffusivities and velocities, making 2fFCS the method of choice for accurately measuring absolute values of these quantities at pico- to nanomolar concentration.

  9. Long Range Surface Plasmon Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Kasry, Amal; Knoll, Wolfgang

    2007-03-01

    Surface plasmon modes, excited at the two sides of a thin metal layer surrounded by two (nearly) identical dielectric media interact via the overlap of their electromagnetic fields. This overlap results in two new-coupled modes, a short and a long-range surface plasmon (LRSP). We demonstrate that combining the LRSP optics with fluorescence spectroscopy can result in a huge enhancement of the fluorescence signal due to the enhanced optical field of the LRSP at the metal dielectric interface, and to its increased evanescent depth into the analyte. This was demonstrated for the detection of the fluorescence intensity of chromophore labeled protein bound to the surface sensor. Beside that, some fundamentals were studied leading to some interesting difference between SPFS and LRSPFS.

  10. Fluorescence spectroscopy for diagnosis of esophageal cancer

    NASA Astrophysics Data System (ADS)

    Panjehpour, Masoud; Overholt, Bergein F.; Vo-Dinh, Tuan; Farris, Christie; Schmidhammer, James L.; Sneed, Rick E.; Buckley, Paul F., III

    1994-07-01

    Laser-induced fluorescence spectroscopy was employed to measure fluorescence emission of normal and malignant tissue during endoscopy in patients with esophageal adenocarcinoma. A nitrogen/dye laser tuned at 410 nm was used for excitation source. The fluorescence lineshape of each spectrum was determined and sampled at 15 nm intervals from 430 nm to 716 nm. A calibration set from normal and malignant spectra were selected. Using stepwise discriminate analysis, significant wavelengths that separated normal and malignant spectra were selected. The intensities at these wavelengths were used to formulate a classification model using linear discriminate analysis. The model was used to classify additional tissue spectra from 26 malignant and 108 normal sites into either normal or malignant spectra with a sensitivity of 100 percent and specificity of 98 percent.

  11. Fluorescence spectroscopy of protein oligomerization in membranes.

    PubMed

    Gorbenko, Galyna P

    2011-05-01

    Fluorescence spectroscopy is one of the most powerful tools for characterization of a multitude of biological processes. Of these, the phenomenon of protein oligomerization attracts especial interest due to its crucial role in the formation of fibrillar protein aggregates (amyloid fibrils) involved in ethiology of so-called protein misfolding diseases. It is becoming increasingly substantiated that protein fibrillization in vivo can be initiated and modulated at membrane-water interface. All steps of membrane-assisted fibrillogenesis, viz., protein adsorption onto lipid bilayer, structural transition of polypeptide chain into a highly aggregation-prone partially folded conformation, assembly of oligomeric nucleus from membrane-bound monomeric species and fiber elongation can be monitored with a mighty family of fluorescence-based techniques. Furthermore, the mechanisms behind cytotoxicity of prefibrillar protein oligomers are highly amenable to fluorescence analysis. The applications of fluorescence spectroscopy to monitoring protein oligomerization in a membrane environment are exemplified and some problems encountered in such kinds of studies are highlighted.

  12. Dye aggregation identified by vibrational coupling using 2D IR spectroscopy

    SciTech Connect

    Oudenhoven, Tracey A.; Laaser, Jennifer E.; Zanni, Martin T.; Joo, Yongho; Gopalan, Padma

    2015-06-07

    We report that a model dye, Re(CO){sub 3}(bypy)CO{sub 2}H, aggregates into clusters on TiO{sub 2} nanoparticles regardless of our preparation conditions. Using two-dimensional infrared (2D IR) spectroscopy, we have identified characteristic frequencies of monomers, dimers, and trimers. A comparison of 2D IR spectra in solution versus those deposited on TiO{sub 2} shows that the propensity to dimerize in solution leads to higher dimer formation on TiO{sub 2}, but that dimers are formed even if there are only monomers in solution. Aggregates cannot be washed off with standard protocols and are present even at submonolayer coverages. We observe cross peaks between aggregates of different sizes, primarily dimers and trimers, indicating that clusters consist of microdomains in close proximity. 2D IR spectroscopy is used to draw these conclusions from measurements of vibrational couplings, but if molecules are close enough to be vibrationally coupled, then they are also likely to be electronically coupled, which could alter charge transfer.

  13. Folding of a heterogeneous β-hairpin peptide from temperature-jump 2D IR spectroscopy

    PubMed Central

    Jones, Kevin C.; Peng, Chunte Sam; Tokmakoff, Andrei

    2013-01-01

    We provide a time- and structure-resolved characterization of the folding of the heterogeneous β-hairpin peptide Tryptophan Zipper 2 (Trpzip2) using 2D IR spectroscopy. The amide I′ vibrations of three Trpzip2 isotopologues are used as a local probe of the midstrand contacts, β-turn, and overall β-sheet content. Our experiments distinguish between a folded state with a type I′ β-turn and a misfolded state with a bulged turn, providing evidence for distinct conformations of the peptide backbone. Transient 2D IR spectroscopy at 45 °C following a laser temperature jump tracks the nanosecond and microsecond kinetics of unfolding and the exchange between conformers. Hydrogen bonds to the peptide backbone are loosened rapidly compared with the 5-ns temperature jump. Subsequently, all relaxation kinetics are characterized by an observed 1.2 ± 0.2-μs exponential. Our time-dependent 2D IR spectra are explained in terms of folding of either native or nonnative contacts from a common compact disordered state. Conversion from the disordered state to the folded state is consistent with a zip-out folding mechanism. PMID:23382249

  14. Amide I'-II' 2D IR spectroscopy provides enhanced protein secondary structural sensitivity.

    PubMed

    Deflores, Lauren P; Ganim, Ziad; Nicodemus, Rebecca A; Tokmakoff, Andrei

    2009-03-11

    We demonstrate how multimode 2D IR spectroscopy of the protein amide I' and II' vibrations can be used to distinguish protein secondary structure. Polarization-dependent amide I'-II' 2D IR experiments on poly-l-lysine in the beta-sheet, alpha-helix, and random coil conformations show that a combination of amide I' and II' diagonal and cross peaks can effectively distinguish between secondary structural content, where amide I' infrared spectroscopy alone cannot. The enhanced sensitivity arises from frequency and amplitude correlations between amide II' and amide I' spectra that reflect the symmetry of secondary structures. 2D IR surfaces are used to parametrize an excitonic model for the amide I'-II' manifold suitable to predict protein amide I'-II' spectra. This model reveals that the dominant vibrational interaction contributing to this sensitivity is a combination of negative amide II'-II' through-bond coupling and amide I'-II' coupling within the peptide unit. The empirically determined amide II'-II' couplings do not significantly vary with secondary structure: -8.5 cm(-1) for the beta sheet, -8.7 cm(-1) for the alpha helix, and -5 cm(-1) for the coil.

  15. [Study on the processing of leech by FTIR and 2D-IR correlation spectroscopy].

    PubMed

    Li, Bing-Ning; Wu, Yan-Wen; Ouyang, Jie; Sun, Su-Qin; Chen, Shun-Cong

    2011-04-01

    The chemical differences of traditional Chinese medicine leech before and after processing were analyzed by FTIR and two-dimensional correlation infrared (2D-IR) spectroscopy. The result showed that the leech was high in protein, with characteristic peaks of amide I, II bands. Comparing the IR spectra of samples, the primary difference was that the characteristic peak of fresh leech was at 1 543 cm(-1), while that of crude and processed leech was at 1 535 cm(-1). A 2D-IR spectrum with heating perturbation was used to track the processing dynamics of leech In the 2D-IR correlation spectra, fresh leech exhibited stronger automatic peaks of the amide I and II bands than that of processed leech, which indicates that the protein components of the fresh leech were more sensitive to heat perturbation than the processed one. Moreover, the result of FTIR and 2D-IR correlation spectra validated that the 3-dimensional structure of protein was damaged and hydrogen bonds were broken after processing, which resulted in the inactivation of protein. The fatty acids and cholesterol components of leech were also oxidized in this process.

  16. Diagnosis of Breast Cancer Using Fluorescence and Reflectance Spectroscopy

    DTIC Science & Technology

    2004-09-01

    breast cancer based on fluorescence and diffuse reflectance spectroscopy . Our first objective include was to characterize the fluorescence properties of...device based on fluorescence and diffuse reflectance spectroscopy has the advantage of being fast, quantitative, and minimally invasive, and has the...Fluorescence and diffuse reflectance spectroscopy in the ultraviolet-visible wavelength range were made with a multi-separation probe at three illumination

  17. The separation of overlapping transitions in β-carotene with broadband 2D electronic spectroscopy

    NASA Astrophysics Data System (ADS)

    Calhoun, Tessa R.; Davis, Jeffrey A.; Graham, Matthew W.; Fleming, Graham R.

    2012-01-01

    Broadband 2D electronic spectroscopy is applied to β-carotene, revealing new insight into the excited state dynamics of carotenoids by exploring the full energetic range encompassing the S0→S2 and S1→S1n transitions at 77 K. Multiple signals are observed in the regime associated with the proposed S∗ state and isolated through separate analysis of rephasing and nonrephasing contributions. Peaks in rephasing pathways display dynamic lineshapes characteristic of coupling to high energy vibrational modes, and simulation with a simple model supports their assignment to impulsive stimulated Raman scattering. A signal persisting beyond 10 ps in the nonrephasing spectra is still under investigation.

  18. Fluorescence Intensity and Polarization from Photodissociated H2, D2 and HD

    NASA Astrophysics Data System (ADS)

    Machacek, J. R.; Andrianarijaona, V. M.; Furst, J. E.; Gay, T. J.; Kilcoyne, A. L. D.; Landers, A. L.; McLaughlin, K. W.

    2009-05-01

    We have measured the intensity and linear polarization of Hα (n=3->n=2) 656.3 nm fluorescence resulting from H and D atoms created by photodissociation of H2, D2 and HD using linearly-polarized photons with energies ranging from 16.5 to 17.6 eV. Between the threshold for atomic n=3 production at 16.6 eV and the n=4 production threshold at 17.3 eV, the relative cross section and polarization data are free from cascade contributions due to higher-lying atomic states. The photon beam energy width used for this work was 3 meV. Comparison of relative intensities to previous measurements [1] show marked differences. However, the polarization is in qualitative agreement. [1] H. Frohlich et al., Z. Phys. D 34, 119 (1995). Support provided by the NSF (Grant PHY-0653379), DOE (LBNL/ALS) and ANSTO (Access to Major Research Facilities Programme).

  19. Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy

    PubMed Central

    Lakowicz, Joseph R.; Ray, Krishanu; Chowdhury, Mustafa; Szmacinski, Henryk; Fu, Yi; Zhang, Jian; Nowaczyk, Kazimierz

    2009-01-01

    Fluorescence spectroscopy is widely used in biological research. Until recently, essentially all fluorescence experiments were performed using optical energy which has radiated to the far-field. By far-field we mean at least several wavelengths from the fluorophore, but propagating far-field radiation is usually detected at larger macroscopic distances from the sample. In recent years there has been a growing interest in the interactions of fluorophores with metallic surfaces or particles. Near-field interactions are those occurring within a wavelength distance of an excited fluorophore. The spectral properties of fluorophores can be dramatically altered by near-field interactions with the electron clouds present in metals. These interactions modify the emission in ways not seen in classical fluorescence experiments. In this review we provide an intuitive description of the complex physics of plasmons and near-field interactions. Additionally, we summarize the recent work on metal–fluorophore interactions and suggest how these effects will result in new classes of experimental procedures, novel probes, bioassays and devices. PMID:18810279

  20. Position-sensitive scanning fluorescence correlation spectroscopy.

    PubMed

    Skinner, Joseph P; Chen, Yan; Müller, Joachim D

    2005-08-01

    Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample. To access the spatial information we synchronize scanning and data acquisition. Synchronization allows us to evaluate correlations for every position along the scanned trajectory. We use a circular scan trajectory in this study. Because the scan radius is constant, the phase angle is sufficient to characterize the position of the beam. We introduce position-sensitive SFCS (PSFCS), where correlations are calculated as a function of lag time and phase. We present the theory of PSFCS and derive expressions for diffusion, diffusion in the presence of flow, and for immobilization. To test PSFCS we compare experimental data with theory. We determine the direction and speed of a flowing dye solution and the position of an immobilized particle. To demonstrate the feasibility of the technique for applications in living cells we present data of enhanced green fluorescent protein measured in the nucleus of COS cells.

  1. Dynamic UltraFast 2D EXchange SpectroscopY (UF-EXSY) of hyperpolarized substrates

    PubMed Central

    Swisher, Christine Leon; Koelsch, Bertram; Sukumar, Subramianam; Sriram, Renuka; Santos, Romelyn Delos; Wang, Zhen Jane; Kurhanewicz, John; Vigneron, Daniel; Larson, Peder

    2015-01-01

    In this work, we present a new ultrafast method for acquiring dynamic 2D EXchange SpectroscopY (EXSY) within a single acquisition. This technique reconstructs two-dimensional EXSY spectra from one-dimensional spectra based on the phase accrual during echo times. The Ultrafast-EXSY acquisition overcomes long acquisition times typically needed to acquire 2D NMR data by utilizing sparsity and phase dependence to dramatically undersample in the indirect time dimension. This allows for the acquisition of the 2D spectrum within a single shot. We have validated this method in simulations and hyperpolarized enzyme assay experiments separating the dehydration of pyruvate and lactate-to-pyruvate conversion. In a renal cell carcinoma cell (RCC) line, bidirectional exchange was observed. This new technique revealed decreased conversion of lactate-to-pyruvate with high expression of monocarboxylate transporter 4 (MCT4), known to correlate with aggressive cancer phenotypes. We also showed feasibility of this technique in vivo in a RCC model where bidirectional exchange was observed for pyruvate–lactate, pyruvate–alanine, and pyruvate–hydrate and were resolved in time. Broadly, the technique is well suited to investigate the dynamics of multiple exchange pathways and applicable to hyperpolarized substrates where chemical exchange has shown great promise across a range of disciplines. PMID:26117655

  2. Probing Spatio-Temporal Correlation in Complex Aqueous Systems through 2D-IR Spectroscopy

    NASA Astrophysics Data System (ADS)

    Bagchi, Biman; Biswas, Rajib; Samanta, Tuhin; Ghosh, Rikhia; Roy, Susmita

    2015-03-01

    Heterogeneity is ubiquitous in aqueous solutions, e.g., in protein and DNA solutions, micelles and reverse micelles, density fluctuations during phase transitions (e,g., water to ice). Origin of heterogeneity can be diverse, sometimes stimulated by external biomolecular subsystems (proteins, DNA, lipids), nanoscopic materials etc, but may also be intrinsic to the thermodynamic nature of the aqueous solution itself. The altered dynamics of water in presence of such diverse surfaces have attracted considerable attention in recent years. However, efficiently capturing the length and timescale of heterogeneous dynamics of water is indeed a challenging task. Recent development of two dimensional infra-red (2D-IR) allows us to estimate length and time scales of such dynamics fairly accurately. In this work, we present a series of interesting studies employing two dimensional infra-red spectroscopy (2D-IR) to investigate (i) dynamics of water inside reverse micelles of varying sizes, (ii) supercritical water near the Widom line that is known to exhibit pronounced density fluctuation and calculate. The respective studies reveal a number of interesting facts. Spatio-temporal correlation of water dynamics with varying size of reverse micelles is well captured through the spectral diffusion of corresponding 2D-IR spectra. In case of supercritical water also, we observe strong signature of dynamic heterogeneity from the elongated nature of the spectra.

  3. Dynamic UltraFast 2D EXchange SpectroscopY (UF-EXSY) of hyperpolarized substrates

    NASA Astrophysics Data System (ADS)

    Leon Swisher, Christine; Koelsch, Bertram; Sukumar, Subramianam; Sriram, Renuka; Santos, Romelyn Delos; Wang, Zhen Jane; Kurhanewicz, John; Vigneron, Daniel; Larson, Peder

    2015-08-01

    In this work, we present a new ultrafast method for acquiring dynamic 2D EXchange SpectroscopY (EXSY) within a single acquisition. This technique reconstructs two-dimensional EXSY spectra from one-dimensional spectra based on the phase accrual during echo times. The Ultrafast-EXSY acquisition overcomes long acquisition times typically needed to acquire 2D NMR data by utilizing sparsity and phase dependence to dramatically undersample in the indirect time dimension. This allows for the acquisition of the 2D spectrum within a single shot. We have validated this method in simulations and hyperpolarized enzyme assay experiments separating the dehydration of pyruvate and lactate-to-pyruvate conversion. In a renal cell carcinoma cell (RCC) line, bidirectional exchange was observed. This new technique revealed decreased conversion of lactate-to-pyruvate with high expression of monocarboxylate transporter 4 (MCT4), known to correlate with aggressive cancer phenotypes. We also showed feasibility of this technique in vivo in a RCC model where bidirectional exchange was observed for pyruvate-lactate, pyruvate-alanine, and pyruvate-hydrate and were resolved in time. Broadly, the technique is well suited to investigate the dynamics of multiple exchange pathways and applicable to hyperpolarized substrates where chemical exchange has shown great promise across a range of disciplines.

  4. Ultrafast Nonlinear Spectroscopy of Red Fluorescent Proteins

    NASA Astrophysics Data System (ADS)

    Konold, Patrick Eugene

    Red-emitting homologues (RFPs) of the native Green Fluorescent Protein (GFP) with emission wavelengths beyond 650 nm are desirable probes for in vivo imaging experiments. They offer the potential for deeper tissue penetration and lower background scatter given a cleaner spectral window. However, bioimaging applications are hindered by poor photophysics ( e.g. low fluorescence quantum yield, high photobleaching), which limits experimental resolution and represents a significant obstacle towards utilization for low copy-number, long-duration imaging applications. In this thesis, a variety of femtosecond nonlinear electronic spectroscopies were employed jointly with site-directed mutagenesis to investigate the photophysical properties of RFPs. In one study, the molecular mechanism of red emission was pursued in two notable RFPs, mPlum and TagRFP675. Solvation dynamics observed with time-resolved transient grating spectroscopy were interpreted with the aid of molecular dynamics simulations to indicate that their red-emission is correlated with the ability of specific chromophore-sidechain hydrogen-bonding interactions to interconvert between direct and water-mediated states. In a second set of studies, two-dimensional double quantum coherence spectroscopy was used to probe the electronic transitions of mPlum. It was discovered that it displayed a response distinctly different from an organic dye in bulk solvent. Modeling indicate of these spectra indicate the spectral features may be attributed to the existence of multiple high-lying (n>1) excited states. The results provide new insight into the electronic structure of these widely used fluorescent probes.

  5. Characterization of Secondary Amide Peptide Bonds Isomerization: Thermodynamics and Kinetics from 2D NMR Spectroscopy

    PubMed Central

    Zhang, Jin; Germann, Markus W.

    2011-01-01

    Secondary amide cis peptide bonds are of even lower abundance than the cis tertiary amide bonds of prolines, yet they are of biochemical importance. Using 2D NMR exchange spectroscopy we investigated the formation of cis peptide bonds in several oligopeptides: Ac-G-G-G-NH2, Ac-I-G-G-NH2, Ac-I-G-G-N-NH2 and its cyclic form: I-G-G-N in DMSO. From the NMR studies, using the amide protons as monitors, an occurrenc.e of 0.13% – 0.23% of cis bonds was obtained at 296 K. The rate constants for the trans to cis conversion determined from 2D EXSY spectroscopy were 4–9·10−3 s−1. Multiple minor conformations were detected for most peptide bonds. From their thermodynamic and kinetic properties the cis isomers are distinguished from minor trans isomers that appear because of an adjacent cis peptide bond. Solvent and sequence effects were investigated utilizing N-methylacetamide and various peptides, which revealed an unique enthalpy profile in DMSO. The cyclization of a tetrapeptide resulted in greatly lowered cis populations and slower isomerization rate compared to its linear counterpart, further highlighting the impact of structural constraints. PMID:21538331

  6. Two-photon-excited fluorescence spectroscopy of atomic fluorine at 170 nm

    NASA Technical Reports Server (NTRS)

    Herring, G. C.; Dyer, Mark J.; Jusinski, Leonard E.; Bischel, William K.

    1988-01-01

    Two-photon-excited fluorescence spectroscopy of atomic fluorine is reported. A doubled dye laser at 286-nm is Raman shifted in H2 to 170 nm (sixth anti-Stokes order) to excite ground-state 2P(0)J fluorine atoms to the 2D(0)J level. The fluorine atoms are detected by one of two methods: observing the fluorescence decay to the 2PJ level or observing F(+) production through the absorption of an additional photon by the excited atoms. Relative two-photon absorption cross sections to and the radiative lifetimes of the 2D(0)J states are measured.

  7. Investigation on the spectral properties of 2D asynchronous fluorescence spectra generated by using variable excitation wavelengths as a perturbation

    NASA Astrophysics Data System (ADS)

    Wang, Jingdan; He, Anqi; Guo, Ran; Wei, Yongju; Feng, Juan; Xu, Yizhuang; Noda, Isao; Wu, Jinguang

    2016-11-01

    Properties of 2D asynchronous spectra generated from a series of fluorescence emission spectra are investigated. Variable excitation wavelengths are utilized as an external perturbation. Based on the results of mathematical analysis and computer simulation, we find that no cross peak will be produced on the 2D asynchronous spectrum if the fluorescent solute under investigation occurs in a single micro-environment. The observation of cross peaks implies that the fluorescent molecule may occur in different micro-environments in a solution. Based on these results, we use 2D asynchronous spectra to investigate the emission spectra of anthracene dissolved in cyclohexane. When the concentration of anthracene is low, no cross peak is produced in the resultant 2D asynchronous spectrum, confirming that anthracene is dissolved as single molecule in the solution. As the concentration elevated, cross peaks appear in the corresponding 2D asynchronous spectra. A plausible explanation of this phenomenon is that anthracene may undergo aggregation via π-π interaction or π-C-H interaction.

  8. Direct Formation of Large-Area 2D Nanosheets from Fluorescent Semiconducting Homopolymer with Orthorhombic Crystalline Orientation.

    PubMed

    Yang, Sanghee; Shin, Suyong; Choi, Inho; Lee, Jaeho; Choi, Tae-Lim

    2017-03-01

    Semiconducting polymers have been widely investigated due to their intriguing optoelectronic properties and their high crystallinity that provides a strong driving force for self-assembly. Although there are various reports of successful self-assembly of nanostructures using semiconducting polymers, direct in situ self-assembly of these polymers into two-dimensional (2D) nanostructures has proven difficult, despite their importance for optoelectronics applications. Here, we report the synthesis of a simple conjugated homopolymer by living cyclopolymerization of a 1,6-heptadiyne (having a fluorene moiety) and its efficient in situ formation of large-area 2D fluorescent semiconducting nanostructures. Using high-resolution imaging tools such as atomic force microscopy and transmission electron microscopy, we observed the solvent-dependent self-assembly behaviors of this homopolymer; the identical starting polymer formed 2D nanosheets with different shapes, such as rectangle, raft, and leaf, when dissolved in different solvents. Furthermore, super-resolution optical microscopy enabled the real-time imaging of the fluorescent 2D nanosheets, revealing their stable and uniform shapes, fluorescence, and solution dynamics. Notably, we propose an orthorhombic crystalline packing model to explain the direct formation of 2D nanostructures based on various diffraction patterns, providing important insight for their shape modulation during the self-assembly.

  9. Fluorescence correlation spectroscopy in semiadhesive wall proximity.

    PubMed

    Sanguigno, Luigi; De Santo, Ilaria; Causa, Filippo; Netti, Paolo A

    2011-11-01

    With examination of diffusion in heterogeneous media through fluorescence correlation spectroscopy, the temporal correlation of the intensity signal shows a long correlation tail and the characteristic diffusion time results are no longer easy to determine. Excluded volume and sticking effects have been proposed to justify such deviations from the standard behavior since all contribute and lead to anomalous diffusion mechanisms . Usually, the anomalous coefficient embodies all the effects of environmental heterogeneity providing too general explanations for the exotic diffusion recorded. Here, we investigated whether the reason of anomalies could be related to a lack of an adequate interpretative model for heterogeneous systems and how the presence of obstacles on the detection volume length scale could affect fluorescence correlation spectroscopy experiments. We report an original modeling of the autocorrelation function where fluorophores experience reflection or adsorption at a wall placed at distances comparable with the detection volume size. We successfully discriminate between steric and adhesion effects through the analysis of long time correlations and evaluate the adhesion strength through the evaluation of probability of being adsorbed and persistence time at the wall on reference data. The proposed model can be readily adopted to gain a better understanding of intracellular and nanoconfined diffusion opening the way for a more rational analysis of the diffusion mechanism in heterogeneous systems and further developing biological and biomedical applications.

  10. Model-based segmentation and quantification of subcellular structures in 2D and 3D fluorescent microscopy images

    NASA Astrophysics Data System (ADS)

    Wörz, Stefan; Heinzer, Stephan; Weiss, Matthias; Rohr, Karl

    2008-03-01

    We introduce a model-based approach for segmenting and quantifying GFP-tagged subcellular structures of the Golgi apparatus in 2D and 3D microscopy images. The approach is based on 2D and 3D intensity models, which are directly fitted to an image within 2D circular or 3D spherical regions-of-interest (ROIs). We also propose automatic approaches for the detection of candidates, for the initialization of the model parameters, and for adapting the size of the ROI used for model fitting. Based on the fitting results, we determine statistical information about the spatial distribution and the total amount of intensity (fluorescence) of the subcellular structures. We demonstrate the applicability of our new approach based on 2D and 3D microscopy images.

  11. APD detectors for biological fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Mazères, S.; Borrel, V.; Magenc, C.; Courrech, J. L.; Bazer-Bachi, R.

    2006-11-01

    Fluorescence spectroscopy is a very convenient and widely used method for studying the molecular background of biological processes [L. Salomé, J.L. Cazeil, A. Lopez, J.F. Tocanne, Eur. Biophys. J. 27 (1998) 391-402]. Chromophores are included in the structure under study and a flash of laser light induces fluorescence (Fluorescence Recovery After Photo-bleaching), the decay of which yields information on the polarity, the speed of rotation, and the speed of diffusion as well as on the temporal and spatial evolution of interactions between molecular species. The method can even be used to study living cells [J.F. Tocanne, L. Cézanne, A. Lopez, Prog. Lipid Res. 33 (1994) 203-237, L. Cezanne, A. Lopez, F. Loste, G. Parnaud, O. Saurel, P. Demange, J.F. Tocanne, Biochemistry 38 (1999) 2779-2786]. This is classically performed with a PM-based system. For biological reasons a decrease of the excitation of the cells is highly desirable. Because the fluorescence response then becomes fainter a significant improvement in detector capability would be welcome. We present here results obtained with an Avalanche Photo Diode (APD)-based system. The small sensitive area of detection allows a very significant improvement in signal/noise ratio, improvement in gain, and the opening-up of a new parameter space. With these new detectors we can begin the study of information transmission between cells through morphine receptors. This work involves both electronics engineers and biophysicists, so results and techniques in both fields will be presented here.

  12. IRIS : A reaction spectroscopy facility with solid H2 /D2 target

    NASA Astrophysics Data System (ADS)

    Holl, Matthias; Kanungo, Ritu; Alcorta, Martin; Andreoiu, Corina; Bidaman, Harris; Burbadge, Christina; Burke, Devin; Chen, Alan; Davids, Barry; Diaz Varela, Alejandra; Garrett, Paul; Hackman, Greg; Ishimoto, Shigeru; Kaur, Satbir; Keefe, Matthew; Kruecken, Reiner; Mansour, Iymad; Randhawa, Jaspreet; Sanetullaev, Alisher; Shotter, Alan; Smith, Jenna; Tanaka, Junki; Tanihata, Isao; Turko, Joseph; Workman, Orry

    2016-09-01

    The charged particle reaction spectroscopy station IRIS at TRIUMF is designed to allow studies of inelastic scattering and transfer reactions for low intensity beams. To do so, a novel solid H2 /D2 target is used in combination with a low pressure ionization chamber for the identification of incoming beam particles. The light ejectiles are measured using a ΔE - E telescope consisting of an annular silicon detector followed by CsI(Tl) array. Another ΔE - E telescope, consisting of two segmented silicon detectors, is used to identify the heavy outgoing particles. An overview of the faciltity will be given and examples from recent experiments that illustrate that facility's capability for reaction studies of exotic nuclei will be shown. Support from Canada Foundation for Innovation, Nova Scotia Research and Innovation Trust and NSERC.

  13. Fast acquisition of high-resolution 2D NMR spectroscopy in inhomogeneous magnetic fields

    NASA Astrophysics Data System (ADS)

    Lin, Liangjie; Wei, Zhiliang; Zeng, Qing; Yang, Jian; Lin, Yanqin; Chen, Zhong

    2016-05-01

    High-resolution nuclear magnetic resonance (NMR) spectroscopy plays an important role in chemical and biological analyses. In this study, we combine the J-coupling coherence transfer module with the echo-train acquisition technique for fast acquisition of high-resolution 2D NMR spectra in magnetic fields with unknown spatial variations. The proposed method shows satisfactory performance on a 5 mM ethyl 3-bromopropionate sample, under a 5-kHz (10 ppm at 11.7 T) B0 inhomogeneous field, as well as under varying degrees of pulse-flip-angle deviations. Moreover, a simulative ex situ NMR measurement is also conducted to show the effectiveness of the proposed pulse sequence.

  14. Rotational Spectroscopy of Vibrationally Excited N_2H^+ and N_2D^+ up to 2 Thz

    NASA Astrophysics Data System (ADS)

    Yu, Shanshan; Pearson, John; Drouin, Brian; Crawford, Timothy J.; Daly, Adam M.; Elliott, Ben; Amano, Takayoshi

    2015-06-01

    Terahertz absorption spectroscopy was employed to extend the measurements on the pure rotational transitions of N_2H^+, N_2D^+ and their 15N-containing isotopologues in the ground state and first excited vibrational states for the three fundamental vibrational modes. In total 88 new pure rotational transitions were observed in the range of 0.7--2.0~THz. The observed transition frequencies were fit to experimental accuracy, and the improved molecular parameters were obtained. The new measurements and predictions will support the analysis of high-resolution astronomical observations made with facilities such as SOFIA and ALMA where laboratory rest frequencies with uncertainties of 1 MHz or smaller are required for proper analysis of velocity resolved astrophysical components.

  15. A 2D correlation Raman spectroscopy analysis of a human cataractous lens

    NASA Astrophysics Data System (ADS)

    Sacharz, Julia; Wesełucha-Birczyńska, Aleksandra; Paluszkiewicz, Czesława; Chaniecki, Piotr; Błażewicz, Marta

    2016-11-01

    This work is a continuation of our study of a cataractous human eye lens removed after phacoemulsification surgery. There are clear differences in the lens colors that allowed for distinguishing two opaque phases in the obtained biological material: the white- and yellow-phase. The Raman spectroscopy and 2D correlation spectroscopy method were used to trace a pathologically altered human cataract lens at a molecular level. Although the Raman spectra of these two phases are relatively similar, taking advantage of 2D correlation, and considering time as an external perturbation, the synchronous and asynchronous spectra were obtained showing completely different patterns. Prominent synchronous auto-peaks appear at 3340, 2920, 1736, 1665 and 1083 cm-1 for the white-, and at 2929 and 1670 cm-1 for the yellow phase. The white phase is characterized by intensive asynchronous peaks at -(2936, 3360), -(1650, 1674) and +(1620,1678). The modifications in the water contained in the white phase structure are ahead of the changes in the protein (CH3-groups), furthermore changes in β-conformation are asynchronous with respect to the α-structure. The yellow phase demonstrates asynchronous peaks: +(2857, 2928), +(1645,1673), +(1663, 1679), and +(1672,1707). These illustrate concomitant modifications in the β- and unordered conformation. Both forms of cataractous human eye lens, white- and yellow-phases, are degenerate forms of the eye lens proteins, both are arranged in a different way. The main differences are observed for the amide I, methyl, methylene and Osbnd H vibrational band region. The effect of Asp, Glu and Tyr amino acids in cataractous lens transformations was observed.

  16. Laser Induced Fluorescence Spectroscopy of Boron Carbide

    NASA Astrophysics Data System (ADS)

    Cheung, A. S.-C.; Ng, Y. W.; Pang, H. F.

    2011-06-01

    Laser induced fluorescence spectrum of boron carbide (BC) between 490 and 560 nm has been recorded and analyzed. Gas-phase BC molecule was produced by the reaction of B2H6 and CH4 in the presence of magnesium atom from laser ablation process. The (0, 0), (1, 0), and (2, 0) bands of the B4 Σ- - X4 Σ- transition were recorded and rotationally analyzed. Spectra of both isotopes: 10BC and 11BC were observed. Equilibrium molecular constants for the B4 Σ- and the X4 Σ- states for both isotopes were determined. A comparison of the determined gas-phase molecular constants with those obtained using matrix isolation spectroscopy and the theoretical calculations will be presented. Financial support from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. HKU 701008P) is gratefully acknowledged.

  17. Glucose Recognition in Vitro Using Fluorescent Spectroscopy

    SciTech Connect

    Noronha, G; Heiss, A M; Reilly, J R; Vachon, Jr, D J; Cary, D R; Zaitseva, N P; Reibold, R A; Lane, S M; Peyser, T A; Satcher, J H

    2001-04-25

    Diabetes is a disease that affects over 16 million people in the USA at a cost of 100 billion dollars annually. The ability to regulate insulin delivery in people with Type 1 diabetes is imperative as is the need to manage glucose levels in all people with this disease. Our current method for monitoring glucose is a (FDA approved) minimally invasive enzymatic sensor that can measure glucose levels in vivo for three days. We are focused on developing a noninvasive implantable glucose sensor that will be interrogated by an external device. The material must be robust, easy to process, biocompatible and resistant to biofouling. In this Presentation we will discuss the development of a new polymeric matrix that can recognize physiological levels of glucose in vitro using fluorescent spectroscopy.

  18. Intraluminal fluorescence spectroscopy catheter with ultrasound guidance

    NASA Astrophysics Data System (ADS)

    Stephens, Douglas N.; Park, Jesung; Sun, Yang; Papaioannou, Thanassis; Marcu, Laura

    2009-05-01

    We demonstrate the feasibility of a time-resolved fluorescence spectroscopy (TRFS) technique for intraluminal investigation of arterial vessel composition under intravascular ultrasound (IVUS) guidance. A prototype 1.8-mm (5.4 Fr) catheter combining a side-viewing optical fiber (SVOF) and an IVUS catheter was constructed and tested with in vitro vessel phantoms. The prototype catheter can locate a fluorophore in the phantom vessel wall, steer the SVOF in place, perform blood flushing under flow conditions, and acquire high-quality TRFS data using 337-nm wavelength excitation. The catheter steering capability used for the coregistration of the IVUS image plane and the SVOF beam produce a guiding precision to an arterial phantom wall site location of 0.53+/-0.16 mm. This new intravascular multimodal catheter enables the potential for in vivo arterial plaque composition identification using TRFS.

  19. Synchronous two-dimensional MIR correlation spectroscopy (2D-COS) as a novel method for screening smoke tainted wine.

    PubMed

    Fudge, Anthea L; Wilkinson, Kerry L; Ristic, Renata; Cozzolino, Daniel

    2013-08-15

    In this study, two-dimensional correlation spectroscopy (2D-COS) combined with mid-infrared (MIR) spectroscopy was evaluated as a novel technique for the identification of spectral regions associated with smoke-affected wine, for the purpose of screening taint arising from grapevine exposure to smoke. Smoke-affected wines obtained from experimental and industry sources were analysed using MIR spectroscopy and chemometrics, and calibration models developed. 2D-COS analysis was used to generate synchronous data maps for red and white cask wines spiked with guaiacol, a marker of smoke taint. Correlations were observed at wavelengths that could be attributable to aromatic C-C stretching, i.e., between 1400 and 1500 cm(-1), indicative of volatile phenols. These results demonstrate the potential of 2D-COS as a rapid, high-throughput technique for the preliminary screening of smoke tainted wine.

  20. Changes and characteristics of dissolved organic matter in a constructed wetland system using fluorescence spectroscopy.

    PubMed

    Yao, Yuan; Li, Yun-Zhen; Guo, Xu-Jing; Huang, Tao; Gao, Ping-Ping; Zhang, Ying-Pei; Yuan, Feng

    2016-06-01

    Domestic wastewater was treated by five constructed wetland beds in series. Dissolved organic matter (DOM) collected from influent and effluent samples from the constructed wetland was investigated using fluorescence spectroscopy combined with fluorescence regional integration (FRI), parallel factor (PARAFAC) analysis, and two-dimensional correlation spectroscopy (2D-COS). This study evaluates the capability of these methods in detecting the spectral characteristics of fluorescent DOM fractions and their changes in constructed wetlands. Fluorescence excitation-emission matrix (EEM) combined with FRI analysis showed that protein-like materials displayed a higher removal ratio compared to humic-like substances. The PARAFAC analysis of wastewater DOM indicated that six fluorescent components, i.e., two protein-like substances (C1 and C6), three humic-like substances (C2, C3 and C5), and one non-humic component (C4), could be identified. Tryptophan-like C1 was the dominant component in the influent DOM. The removal ratios of six fluorescent components (C1-C6) were 56.21, 32.05, 49.19, 39.90, 29.60, and 45.87 %, respectively, after the constructed wetland treatment. Furthermore, 2D-COS demonstrated that the sequencing of spectral changes for fluorescent DOM followed the order 298 nm → 403 nm → 283 nm (310-360 nm) in the constructed wetland, suggesting that the peak at 298 nm is associated with preferential tryptophan fluorescence removal. Variation of the fluorescence index (FI) and the ratio of fluorescence components indicated that the constructed wetland treatment resulted in the decrease of fluorescent organic pollutant with increasing the humification and chemical stability of the DOM.

  1. Anharmonic vibrational modes of nucleic acid bases revealed by 2D IR spectroscopy.

    PubMed

    Peng, Chunte Sam; Jones, Kevin C; Tokmakoff, Andrei

    2011-10-05

    Polarization-dependent two-dimensional infrared (2D IR) spectra of the purine and pyrimadine base vibrations of five nucleotide monophosphates (NMPs) were acquired in D(2)O at neutral pH in the frequency range 1500-1700 cm(-1). The distinctive cross-peaks between the ring deformations and carbonyl stretches of NMPs indicate that these vibrational modes are highly coupled, in contrast with the traditional peak assignment, which is based on a simple local mode picture such as C═O, C═N, and C═C double bond stretches. A model of multiple anharmonically coupled oscillators was employed to characterize the transition energies, vibrational anharmonicities and couplings, and transition dipole strengths and orientations. No simple or intuitive structural correlations are found to readily assign the spectral features, except in the case of guanine and cytosine, which contain a single local CO stretching mode. To help interpret the nature of these vibrational modes, we performed density functional theory (DFT) calculations and found that multiple ring vibrations are coupled and delocalized over the purine and pyrimidine rings. Generally, there is close correspondence between the experimental and computational results, provided that the DFT calculations include explicit waters solvating hydrogen-bonding sites. These results provide direct experimental evidence of the delocalized nature of the nucleotide base vibrations via a nonperturbative fashion and will serve as building blocks for constructing a structure-based model of DNA and RNA vibrational spectroscopy.

  2. Characterization and quantification of humic substances 2D-Fluorescence by usage of extended size exclusion chromatography.

    PubMed

    Wagner, Martin; Schmidt, Wido; Imhof, Lutz; Grübel, Anika; Jähn, Camilla; Georgi, Denise; Petzoldt, Heike

    2016-04-15

    In this article, two methods for in-depth analysis of humic substances fluorescence are presented. The first one allows the combined analysis of fluorescence excitation-emission matrix (EEM) with chromatography technique. The main issue is the coupling of size exclusion chromatography (SEC) with spectroscopy by the use of an absorption and a fluorescence spectrometer as additional detectors. These allow a detailed characterization of humic substances depending on their molecular size, concentration and optical properties. For the evaluation of the resulting complex data, a model based on non-negative matrix factorization, which is also presented in this article, was developed. From the results of the examined humic substances standards, the second method was developed. It allows the characterization and quantification of humic substances fluorescence of a natural water sample solely on the basis of an excitation-emission matrix. The validation of the model is carried out within the framework of extensive analysis of real water samples.

  3. Local conformations and excited state dynamics of porphyrins and nucleic acids by 2-dimensional fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Widom, Julia R.

    Biological systems present many challenges to researchers attempting to study them using spectroscopy. Low specificity, low sensitivity, and broad and overlapping lineshapes limit the amount of information that can be obtained in experiments. Two-dimensional fluorescence spectroscopy (2D FS) is a highly sensitive and information-rich spectroscopic technique that was developed to study the conformations and excited state dynamics of systems exhibiting exciton coupling. In this dissertation, I describe a variety of extensions of 2D FS that further increase its utility for the study of biological systems. I describe experiments on a dimer of zinc tetraphenylporphyrin embedded in a membrane, in which the signals from two conformational subpopulations were separated in order to study the thermodynamics of their interconversion. I present proof-of-principle experiments on nucleic acids that utilize fluorescence resonance energy transfer to separate signals from different subpopulations. I also describe experiments in which 2D FS was performed using ultraviolet excitation to determine the conformation of a dinucleotide of a fluorescent analogue of the nucleic acid base adenine. I discuss experiments on porphyrin dimers in which 2D FS was used as a probe of excited state dynamics. Finally, I present model calculations for a proposed variation of 2D FS in which entangled photons would be used as the excitation source. These calculations suggest that this approach has the potential to yield significantly narrower spectral lineshapes than conventional 2D FS. These experiments and calculations yield new insight into the systems investigated and establish a `toolbox' of variations of 2D FS that can be used to gain as much information as possible from experiments on challenging systems such as protein-DNA complexes.

  4. [Analysis of Three Polycyclic Aromatic Hydrocarbons in Solution Based on Two-Dimensional Fluorescence Correlation Spectroscopy].

    PubMed

    Zhou, Chang-hong; Zhao, Mei-rong; Yang, Ren-jie; Zhu, Wen-bi; Dong, Gui-mei

    2016-02-01

    Polycyclic aromatic hydrocarbons (PAHs) are listed as the priority pollutants. It is difficult to resolve effectively the peaks of PAHs by conventional one-dimensional fluorescence spectroscopy due to its low content and the overlapping fluorescence three mixed ystems and a total of 27 samples, are to be prepared with different concentrations of three PAHs. Concentrations of three PAHS are monotonically increasing or decreasing in each mixed system. Then the 2D fluorescence correlation spectrum of each mixed systems will be calculated under the perturbation of the concentration of anthracene, phenanthrene and pyrene in solution. There are seven strong autopeaks at 425, 402, 381, 373, 365, 393 and 347 nm in synchronous 2D correlation spectrum. The fluorescence peak of phenanthrene at 347 nm is uncovered in three mixed systems, so the band at 347 nm is to be used as clues for further assignment. According to positive or negative cross peaks at 347 nm in synchronous 2D correlation spectrum, we can know that the peaks at 402, 381, 425 and 452 nm are assigned to anthracene, the peaks at 373 and 393 nm are assigned to pyrene, and the peaks at 365, 356 and 347 nm are assigned to phenanthrene. The fluorescence peak of phenanthrene at 385 nm is shown in asynchronous 2D correlation spectrum; it means the spectral resolution of asynchronous spectrum is better than the synchronous spectrum. The results are that it is feasible to analyze serious overlapping multi-component PAHs using two-dimensional fluorescence correlation spectroscopy, which can be extended to the detection of other pollutants in the air.

  5. Solution conformation of 2-aminopurine dinucleotide determined by ultraviolet two-dimensional fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Widom, Julia R.; Johnson, Neil P.; von Hippel, Peter H.; Marcus, Andrew H.

    2013-02-01

    We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analogue of the nucleic acid base adenine. This was accomplished by extending two-dimensional fluorescence spectroscopy (2D FS)—a fluorescence-detected variation of 2D electronic spectroscopy—to excite molecular transitions in the ultraviolet (UV) regime. A collinear sequence of four ultrafast laser pulses centered at 323 nm was used to resonantly excite the coupled transitions of 2-AP dinucleotide. The phases of the optical pulses were continuously swept at kilohertz frequencies, and the ensuing nonlinear fluorescence was phase-synchronously detected at 370 nm. Upon optimization of a point-dipole coupling model to our data, we found that in aqueous buffer the 2-AP dinucleotide adopts an average conformation in which the purine bases are non-helically stacked (center-to-center distance R12 = 3.5 ± 0.5 Å , twist angle θ12 = 5° ± 5° ), which differs from the conformation of such adjacent bases in duplex DNA. These experiments establish UV-2D FS as a method for examining the local conformations of an adjacent pair of fluorescent nucleotides substituted into specific DNA or RNA constructs, which will serve as a powerful probe to interpret, in structural terms, biologically significant local conformational changes within the nucleic acid framework of protein-nucleic acid complexes.

  6. 2D ratiometric fluorescent pH sensor for tracking of cells proliferation and metabolism.

    PubMed

    Ma, Jun; Ding, Changqin; Zhou, Jie; Tian, Yang

    2015-08-15

    Extracellular pH plays a vital role no matter in physiological or pathological studies. In this work, a hydrogel, CD@Nile-FITC@Gel (Gel sensor), entrapping the ratiometric fluorescent probe CD@Nile-FITC was developed. The Gel sensor was successfully used for real-time extracellular pH monitoring. In the case of CD@Nile-FITC, pH-sensitive fluorescent dye fluorescein isothiocyanate (FITC) was chosen as the response signal for H(+) and Nile blue chloride (Nile) as the reference signal. The developed fluorescent probe exhibited high selectivity for pH over other metal ions and amino acids. Meanwhile, the carbon-dots-based inorganic-organic probe demonstrated excellent photostability against long-term light illumination. In order to study the extracellular pH change in processes of cell proliferation and metabolism, CD@Nile-FITC probe was entrapped in sodium alginate gel and consequently formed CD@Nile-FITC@Gel. The MTT assay showed low cytotoxicity of the Gel and the pH titration indicated that it could monitor the pH fluctuations linearly and rapidly within the pH range of 6.0-9.0, which is valuable for physiological pH determination. As expected, the real-time bioimaging of the probe was successfully achieved.

  7. Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy

    ERIC Educational Resources Information Center

    Healy, Eamonn F.

    2007-01-01

    The effective use of fluorescence spectroscopy for determining the binding of the intercalcating agent crhidium bromide to DNA is being described. The analysis used simple measurement techniques and hence can be easily adopted by the students for a better understanding.

  8. Fluorescence spectroscopy for rapid detection and classification of bacterial pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study deals with the rapid detection and classification of three bacteria, Escherichia coli, Salmonella, and Campylobacter, using fluorescence spectroscopy and multivariative analysis. Each bacterial sample was diluted in physiologic saline for analysis. Fluoroscence spectra were collected ...

  9. Native fluorescence spectroscopy of thymus and fat tissues

    NASA Astrophysics Data System (ADS)

    Tang, Gui C.; Oz, Mehmet C.; Reid, V.; Steinglass, K.; Ginsberg, Mark D.; Jacobowitz, Larry; Alfano, Robert R.

    1993-08-01

    Fluorescence spectroscopy of the human thymus gland and surrounding mediastinal fat were measured to evaluate this approach in distinguishing between thymus and fat tissues during therapeutic surgery for myasthenia gravis disease.

  10. An algorithm to correct 2D near-infrared fluorescence signals using 3D intravascular ultrasound architectural information

    NASA Astrophysics Data System (ADS)

    Mallas, Georgios; Brooks, Dana H.; Rosenthal, Amir; Vinegoni, Claudio; Calfon, Marcella A.; Razansky, R. Nika; Jaffer, Farouc A.; Ntziachristos, Vasilis

    2011-03-01

    Intravascular Near-Infrared Fluorescence (NIRF) imaging is a promising imaging modality to image vessel biology and high-risk plaques in vivo. We have developed a NIRF fiber optic catheter and have presented the ability to image atherosclerotic plaques in vivo, using appropriate NIR fluorescent probes. Our catheter consists of a 100/140 μm core/clad diameter housed in polyethylene tubing, emitting NIR laser light at a 90 degree angle compared to the fiber's axis. The system utilizes a rotational and a translational motor for true 2D imaging and operates in conjunction with a coaxial intravascular ultrasound (IVUS) device. IVUS datasets provide 3D images of the internal structure of arteries and are used in our system for anatomical mapping. Using the IVUS images, we are building an accurate hybrid fluorescence-IVUS data inversion scheme that takes into account photon propagation through the blood filled lumen. This hybrid imaging approach can then correct for the non-linear dependence of light intensity on the distance of the fluorescence region from the fiber tip, leading to quantitative imaging. The experimental and algorithmic developments will be presented and the effectiveness of the algorithm showcased with experimental results in both saline and blood-like preparations. The combined structural and molecular information obtained from these two imaging modalities are positioned to enable the accurate diagnosis of biologically high-risk atherosclerotic plaques in the coronary arteries that are responsible for heart attacks.

  11. Phase-Resolved Heterodyne-Detected Transient Grating Enhances the Capabilities of 2D IR Echo Spectroscopy.

    PubMed

    Jin, Geun Young; Kim, Yung Sam

    2017-02-09

    2D IR echo spectroscopy, with high sensitivity and femtosecond time resolution, enables us to understand structure and ultrafast dynamics of molecular systems. Application of this experimental technique on weakly absorbing samples, however, had been limited by the precise and unambiguous phase determination of the echo signals. In this study, we propose a new experimental scheme that significantly increases the phase stability of the involved IR pulses. We have demonstrated that the incorporation of phase-resolved heterodyne-detected transient grating (PR-HDTG) spectroscopy greatly enhances the capabilities of 2D IR spectroscopy. The new experimental scheme has been used to obtain 2D IR spectra on weakly absorbing azide ions (N3(-)) in H2O (absorbance ∼0.025), free of phase ambiguity even at large waiting times. We report the estimated spectral diffusion time scale (1.056 ps) of azide ions in aqueous solution from the 2D IR spectra and the vibrational lifetime (750 ± 3 fs) and the reorientation time (1108 ± 24 fs) from the PR-HDTG spectra.

  12. Multiphoton excited fluorescence spectroscopy of biomolecular systems

    NASA Astrophysics Data System (ADS)

    Birch, David J. S.

    2001-09-01

    Recent work on the emerging application of multiphoton excitation to fluorescence studies of biomolecular dynamics and structure is reviewed. The fundamental principles and experimental techniques of multiphoton excitation are outlined, fluorescence lifetimes, anisotropy and spectra in membranes, proteins, hydrocarbons, skin, tissue and metabolites are featured, and future opportunities are highlighted.

  13. Speckle spectroscopy of fluorescent randomly inhomogeneous media

    NASA Astrophysics Data System (ADS)

    Zimnyakov, D. A.; Asharchuk, I. A.; Yuvchenko, S. A.; Sviridov, A. P.

    2016-11-01

    We propose a coherence optical method for probing fluorescent randomly inhomogeneous media based on the statistical analysis of spatial fluctuations of spectrally selected fluorescence radiation. We develop a phenomenological model that interrelates the flicker index of the spatial distribution of the fluorescence intensity at a fixed wavelength and the mean path difference of partial components of the fluorescence radiation field in the probed medium. The results of experimental approbation of the developed method using the layers of densely packed silicon dioxide particles saturated with the aqueous rhodamine 6G solution with a high concentration of the dye are presented. The experimentally observed significant decrease in the flicker index under the wavelength tuning from the edges of the fluorescence spectrum towards it central part is presumably a manifestation of spectrally dependent negative absorption in the medium.

  14. Fluorescence correlation spectroscopy using quantum dots: advances, challenges and opportunities.

    PubMed

    Heuff, Romey F; Swift, Jody L; Cramb, David T

    2007-04-28

    Semiconductor nanocrystals (quantum dots) have been increasingly employed in measuring the dynamic behavior of biomacromolecules using fluorescence correlation spectroscopy. This poses a challenge, because quantum dots display their own dynamic behavior in the form of intermittent photoluminescence, also known as blinking. In this review, the manifestation of blinking in correlation spectroscopy will be explored, preceded by an examination of quantum dot blinking in general.

  15. 2D laser-collision induced fluorescence in low-pressure argon discharges

    SciTech Connect

    Barnat, E. V.; Weatherford, B. R.

    2015-09-25

    Development and application of laser-collision induced fluorescence (LCIF) diagnostic technique is presented for the use of interrogating argon plasma discharges. Key atomic states of argon utilized for the LCIF method are identified. A simplified two-state collisional radiative model is then used to establish scaling relations between the LCIF, electron density, and reduced electric fields (E/N). The procedure used to generate, detect and calibrate the LCIF in controlled plasma environments is discussed in detail. LCIF emanating from an argon discharge is then presented for electron densities spanning 109 e cm–3 to 1012 e cm–3 and reduced electric fields spanning 0.1 Td to 40 Td. Lastly, application of the LCIF technique for measuring the spatial distribution of both electron densities and reduced electric field is demonstrated.

  16. 2D laser-collision induced fluorescence in low-pressure argon discharges

    DOE PAGES

    Barnat, E. V.; Weatherford, B. R.

    2015-09-25

    Development and application of laser-collision induced fluorescence (LCIF) diagnostic technique is presented for the use of interrogating argon plasma discharges. Key atomic states of argon utilized for the LCIF method are identified. A simplified two-state collisional radiative model is then used to establish scaling relations between the LCIF, electron density, and reduced electric fields (E/N). The procedure used to generate, detect and calibrate the LCIF in controlled plasma environments is discussed in detail. LCIF emanating from an argon discharge is then presented for electron densities spanning 109 e cm–3 to 1012 e cm–3 and reduced electric fields spanning 0.1 Tdmore » to 40 Td. Lastly, application of the LCIF technique for measuring the spatial distribution of both electron densities and reduced electric field is demonstrated.« less

  17. Fluorescence Correlation Spectroscopy: A Review of Biochemical and Microfluidic Applications

    PubMed Central

    Tian, Yu; Martinez, Michelle M.

    2011-01-01

    Over the years fluorescence correlation spectroscopy (FCS) has proven to be a useful technique that has been utilized in several fields of study. Although FCS initially suffered from poor signal to noise ratios, the incorporation of confocal microscopy has overcome this drawback and transformed FCS into a sensitive technique with high figures of merit. In addition, tandem methods have evolved to include dual-color cross-correlation, total internal reflection fluorescence correlation, and fluorescence lifetime correlation spectroscopy combined with time-correlated single photon counting. In this review, we discuss several applications of FSC for biochemical, microfluidic, and cellular investigations. PMID:21396180

  18. Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

    PubMed Central

    Paredes, Jose M.; Casares, Salvador; Ruedas-Rama, Maria J.; Fernandez, Elena; Castello, Fabio; Varela, Lorena; Orte, Angel

    2012-01-01

    Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies. PMID:22949804

  19. Laser-induced fluorescence spectroscopy at endoscopy

    NASA Astrophysics Data System (ADS)

    Qu, Jianan Y.; MacAulay, Calum E.; Lam, Stephen; Palcic, Branko

    1994-07-01

    A spectrofluorometry system has been developed for the collection of laser induced fluorescense spectra of tissue during endoscopy. In this system, a catheter with seven optical fibers was used to deliver the excitation light and collect the emitted fluorescence. The system enables one to switch from regular endoscopy into fluorescence measurement in 50 ms using a computerized shutter system. The fluorescence spectra can be recorded in 100 ms. This spectrofluorometry system has been used to obtain spectra from bronchial, larynx and nasopharyngeal tissues when employed with the appropriate endoscopes. The results demonstrate that laser induced fluorescence can be used to differentiate abnormal tissue from normal tissue. The illumination and fluorescence collection patterns of this system have been modeled using a Monte Carlo simulation. The Monte Carlo simulation data shows that the spectra recorded by our collection pattern is very close to the intrinsic spectra of tissue. The experimental results and the Monte Carlo simulation suggest that changes in fluorescence intensity are more robust for the detection of early cancers than the differences in spectral characteristics.

  20. Probing interband coulomb interactions in semiconductor nanostructures with 2D double-quantum coherence spectroscopy.

    PubMed

    Velizhanin, Kirill A; Piryatinski, Andrei

    2011-05-12

    Employing the interband exciton scattering model, we have derived a closed set of equations determining the 2D double-quantum coherence signal sensitive to the interband Coulomb interactions (i.e., many-body Coulomb interactions leading to the couplings between exciton and biexciton bands) in semiconductor nanostructures such as nanocrystals, quantum wires, wells, and carbon nanotubes. Our general analysis of 2D double-quantum coherence resonances has demonstrated that the interband Coulomb interactions lead to new cross-peaks whose appearance can be interpreted as a result of exciton and biexciton state mixing. The presence of the strongly coupled resonant states and weakly coupled background of off-resonant states can significantly simplify cross-peak analysis by eliminating the congested background spectrum. Our simulations of the 2D double-quantum coherence signal in PbSe NCs have validated this approach.

  1. Noise reduction methods applied to two-dimensional correlation spectroscopy (2D-COS) reveal complementary benefits of pre- and post-treatment.

    PubMed

    Foist, Rod B; Schulze, H Georg; Ivanov, Andre; Turner, Robin F B

    2011-05-01

    Two-dimensional correlation spectroscopy (2D-COS) is a powerful spectral analysis technique widely used in many fields of spectroscopy because it can reveal spectral information in complex systems that is not readily evident in the original spectral data alone. However, noise may severely distort the information and thus limit the technique's usefulness. Consequently, noise reduction is often performed before implementing 2D-COS. In general, this is implemented using one-dimensional (1D) methods applied to the individual input spectra, but, because 2D-COS is based on sets of successive spectra and produces 2D outputs, there is also scope for the utilization of 2D noise-reduction methods. Furthermore, 2D noise reduction can be applied either to the original set of spectra before performing 2D-COS ("pretreatment") or on the 2D-COS output ("post-treatment"). Very little work has been done on post-treatment; hence, the relative advantages of these two approaches are unclear. In this work we compare the noise-reduction performance on 2D-COS of pretreatment and post-treatment using 1D (wavelets) and 2D algorithms (wavelets, matrix maximum entropy). The 2D methods generally outperformed the 1D method in pretreatment noise reduction. 2D post-treatment in some cases was superior to pretreatment and, unexpectedly, also provided correlation coefficient maps that were similar to 2D correlation spectroscopy maps but with apparent better contrast.

  2. 2D-Raman-THz spectroscopy: A sensitive test of polarizable water models

    NASA Astrophysics Data System (ADS)

    Hamm, Peter

    2014-11-01

    In a recent paper, the experimental 2D-Raman-THz response of liquid water at ambient conditions has been presented [J. Savolainen, S. Ahmed, and P. Hamm, Proc. Natl. Acad. Sci. U. S. A. 110, 20402 (2013)]. Here, all-atom molecular dynamics simulations are performed with the goal to reproduce the experimental results. To that end, the molecular response functions are calculated in a first step, and are then convoluted with the laser pulses in order to enable a direct comparison with the experimental results. The molecular dynamics simulation are performed with several different water models: TIP4P/2005, SWM4-NDP, and TL4P. As polarizability is essential to describe the 2D-Raman-THz response, the TIP4P/2005 water molecules are amended with either an isotropic or a anisotropic polarizability a posteriori after the molecular dynamics simulation. In contrast, SWM4-NDP and TL4P are intrinsically polarizable, and hence the 2D-Raman-THz response can be calculated in a self-consistent way, using the same force field as during the molecular dynamics simulation. It is found that the 2D-Raman-THz response depends extremely sensitively on details of the water model, and in particular on details of the description of polarizability. Despite the limited time resolution of the experiment, it could easily distinguish between various water models. Albeit not perfect, the overall best agreement with the experimental data is obtained for the TL4P water model.

  3. 2D-Raman-THz spectroscopy: A sensitive test of polarizable water models

    SciTech Connect

    Hamm, Peter

    2014-11-14

    In a recent paper, the experimental 2D-Raman-THz response of liquid water at ambient conditions has been presented [J. Savolainen, S. Ahmed, and P. Hamm, Proc. Natl. Acad. Sci. U. S. A. 110, 20402 (2013)]. Here, all-atom molecular dynamics simulations are performed with the goal to reproduce the experimental results. To that end, the molecular response functions are calculated in a first step, and are then convoluted with the laser pulses in order to enable a direct comparison with the experimental results. The molecular dynamics simulation are performed with several different water models: TIP4P/2005, SWM4-NDP, and TL4P. As polarizability is essential to describe the 2D-Raman-THz response, the TIP4P/2005 water molecules are amended with either an isotropic or a anisotropic polarizability a posteriori after the molecular dynamics simulation. In contrast, SWM4-NDP and TL4P are intrinsically polarizable, and hence the 2D-Raman-THz response can be calculated in a self-consistent way, using the same force field as during the molecular dynamics simulation. It is found that the 2D-Raman-THz response depends extremely sensitively on details of the water model, and in particular on details of the description of polarizability. Despite the limited time resolution of the experiment, it could easily distinguish between various water models. Albeit not perfect, the overall best agreement with the experimental data is obtained for the TL4P water model.

  4. In vivo 1D and 2D correlation MR spectroscopy of the soleus muscle at 7T

    NASA Astrophysics Data System (ADS)

    Ramadan, Saadallah; Ratai, Eva-Maria; Wald, Lawrence L.; Mountford, Carolyn E.

    2010-05-01

    AimThis study aims to (1) undertake and analyse 1D and 2D MR correlation spectroscopy from human soleus muscle in vivo at 7T, and (2) determine T1 and T2 relaxation time constants at 7T field strength due to their importance in sequence design and spectral quantitation. MethodSix healthy, male volunteers were consented and scanned on a 7T whole-body scanner (Siemens AG, Erlangen, Germany). Experiments were undertaken using a 28 cm diameter detunable birdcage coil for signal excitation and an 8.5 cm diameter surface coil for signal reception. The relaxation time constants, T1 and T2 were recorded using a STEAM sequence, using the 'progressive saturation' method for the T1 and multiple echo times for T2. The 2D L-Correlated SpectroscopY (L-COSY) method was employed with 64 increments (0.4 ms increment size) and eight averages per scan, with a total time of 17 min. ResultsT1 and T2 values for the metabolites of interest were determined. The L-COSY spectra obtained from the soleus muscle provided information on lipid content and chemical structure not available, in vivo, at lower field strengths. All molecular fragments within multiple lipid compartments were chemically shifted by 0.20-0.26 ppm at this field strength. 1D and 2D L-COSY spectra were assigned and proton connectivities were confirmed with the 2D method. ConclusionIn vivo 1D and 2D spectroscopic examination of muscle can be successfully recorded at 7T and is now available to assess lipid alterations as well as other metabolites present with disease. T1 and T2 values were also determined in soleus muscle of male healthy volunteers.

  5. Time-resolved spectroscopy of the fluorescence quenching of a donor — acceptor pair by halothane

    NASA Astrophysics Data System (ADS)

    Sharma, A.; Draxler, S.; Lippitsch, M. E.

    1992-04-01

    Donor (anthracene) sensitized acceptor (perylene) fluorescence is quenched more efficiently by halothane than is intrinsic perylene fluorescence. The underlying process of dynamic fluorescence quenching is investigated by time-resolved fluorescence spectroscopy.

  6. Ultrafast slaving dynamics at the protein-water interface studied with 2D-IR spectroscopy

    NASA Astrophysics Data System (ADS)

    King, J. T.; Kubarych, K. J.

    2013-03-01

    The dynamics of hen egg white lysozyme in D2O/glycerol mixtures is studied using two-dimensional infrared spectroscopy. The hydration dynamics and the protein dynamics are studied simultaneously through vibrational probes attached to the protein surface.

  7. Recovering the Fermi surface with 2D-ACAR spectroscopy in samples with defects

    NASA Astrophysics Data System (ADS)

    Dugdale, S. B.; Laverock, J.

    2014-04-01

    When two-dimensional angular correlation of positron annihilation radiation (2D-ACAR) experiments are performed in metals containing defects, conventional analysis in which the measured momentum distribution is folded back into the first Brillouin zone is rendered ineffective due to the contribution from positrons annihilating from the defect. However, by working with the radial anisotropy of the spectrum, it is shown that an image of the Fermi surface can be recovered since the defect contribution is essentially isotropic.

  8. Interrogating Fiber Formation Kinetics with Automated 2D-IR Spectroscopy

    NASA Astrophysics Data System (ADS)

    Strasfeld, David B.; Ling, Yun L.; Shim, Sang-Hee; Zanni, Martin T.

    A new method for collecting 2D-IR spectra that utilizes both a pump-probe beam geometry and a mid-IR pulse shaper is used to gain a fuller understanding of fiber formation in the human islet amyloid polypeptide (hIAPP). We extract structural kinetics in order to better understand aggregation in hIAPP, the protein component of the amyloid fibers found to inhibit insulin production in type II diabetes patients.

  9. Laser-induced fluorescence spectroscopy in tissue local necrosis detection

    NASA Astrophysics Data System (ADS)

    Cip, Ondrej; Buchta, Zdenek; Lesundak, Adam; Randula, Antonin; Mikel, Bretislav; Lazar, Josef; Veverkova, Lenka

    2014-03-01

    The recent effort leads to reliable imaging techniques which can help to a surgeon during operations. The fluorescence spectroscopy was selected as very useful online in vivo imaging method to organics and biological materials analysis. The presented work scopes to a laser induced fluorescence spectroscopy technique to detect tissue local necrosis in small intestine surgery. In first experiments, we tested tissue auto-fluorescence technique but a signal-to-noise ratio didn't express significant results. Then we applied a contrast dye - IndoCyanine Green (ICG) which absorbs and emits wavelengths in the near IR. We arranged the pilot experimental setup based on highly coherent extended cavity diode laser (ECDL) used for stimulating of some critical areas of the small intestine tissue with injected ICG dye. We demonstrated the distribution of the ICG exciter with the first file of shots of small intestine tissue of a rabbit that was captured by high sensitivity fluorescent cam.

  10. Fluorescent Lifetime Spectroscopy in Random Media

    NASA Astrophysics Data System (ADS)

    Hutchinson, Christina Laura

    Recently, an abundance of near-infrared phosphorescent and fluorescent probes have been developed whose lifetime is sensitive to changes in the local environment. The lifetime of these probes can be readily determined in a dilute, non-scattering media using conventional time- and frequency-domain techniques. From the lifetime, the concentration of metabolites can be found using the Stern-Volmer relationship. However, in highly scattering media such as tissues and particulate process streams, measurement of lifetime is complicated by the time delay associated with light scatter. In this dissertation, frequency-domain measurements of photon migration are developed for measuring fluorescent lifetimes independent of absorption and scattering properties of tissues and other random media. The measurement consists of launching, onto the surface of the medium, excitation light whose intensity is sinusoidally modulated at megahertz frequencies. The fluorescent light generated within the medium is intensity modulated at the same frequency, but phase-shifted and amplitude demodulated relative to the incident excitation source. In addition, the excitation light is also phase-shifted and amplitude demodulated relative to the incident excitation source. From Green's function analysis, finite element computations, and experimental measurements of fluorescent phase-shift and amplitude demodulation, we show it is possible to determine fluorophore lifetime of common laser dyes in a tissue mimicking phantom. Furthermore, the finite element computations of excitation and fluorescent light fluence show that when the fluorophore is uniformly distributed within a medium, signals re-emitted at the surface do not originate from significant depths if its lifetime is greater than the photon migration time associated with scatter. Consequently, this research points to the development of short-lived fluorescent compounds for biodiagnostics using properly referenced frequency

  11. Spectroscopic-tomography of biological membrane with high-spatial resolution by the imaging-type 2D Fourier spectroscopy

    NASA Astrophysics Data System (ADS)

    Inui, Asuka; Tsutsumi, Ryosuke; Qi, Wei; Takuma, Takashi; Ishimaru, Ichirou

    2011-07-01

    We proposed the imaging-type 2-dimensional Fourier spectroscopy that is the phase-shift interferometry between the objective lights. The proposed method can measure the 2D spectral image at the limited depth. Because of the imaging optical system, the 2D spectral images can be measured in high spatial resolution. And in the depth direction, we can get the spectral distribution only in the focal plane. In this report, we mention about the principle of the proposed wide field imaging-type 2D Fourier spectroscopy. And, we obtained the spectroscopic tomography of biological tissue of mouse's ear. In the visible region, we confirmed the difference of spectral characteristics between blood vessel region and other region. In the near infrared region (λ=900nm~1700nm), we can obtain the high-contrast blood vessel image of mouse's ear in the deeper part by InGaAs camera. Furthermore, in the middle infrared region(λ=8μ~14μm), we have successfully measured the radiation spectroscopic-imaging with wild field of view by the infrared module, such as the house plants. Additionally, we propose correction geometrical model that can convert the mechanical phase-shift value into the substantial phase difference in each oblique optical axes. We successfully verified the effectiveness of the proposed correction geometrical model and can reduce the spectral error into the error range into +/-3nm using the He-Ne laser whose wavelength 632.8nm.

  12. Combined fiber probe for fluorescence lifetime and Raman spectroscopy

    PubMed Central

    Dochow, Sebastian; Ma, Dinglong; Latka, Ines; Bocklitz, Thomas; Hartl, Brad; Bec, Julien; Fatakdawala, Hussain; Marple, Eric; Urmey, Kirk; Wachsmann-Hogiu, Sebastian; Schmitt, Michael; Marcu, Laura; Popp, Jürgen

    2016-01-01

    In this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. PMID:26093843

  13. Two-Dimensional Fluorescence Difference Spectroscopy to Characterize Nanoparticles and their Interactions.

    PubMed

    Hurst, Miranda N; DeLong, Robert K

    2016-09-14

    Two dimensional fluorescence difference spectroscopy (2D FDS) detects nanoparticle interactions following surface functionalization and biomolecule loading by generating a spectral signature of the fluorescent intensity per excitation and emission wavelengths. Comparing metal oxide nanoparticles revealed a unique spectral signature per material composition. 2D FDS showed to be sensitive to changes in surface properties between ZnO NPs synthesized by different methods. ZnO NP loaded with glycol chitosan, polyacrylic acid (PAA), or methoxy polyethylene glycol (mPEG) exhibited a distinct spectral signature shift. ZnO NP loaded with Torula Yeast RNA (TYRNA)(640 nm), polyinosinic: polycytidylic acid (pIC)(680 nm), or splice switching oligonucleotide (SSO)(650 nm) each revealed a shift in emission. Ras-Binding domain (RBD) at three concentrations (25, 37.5, 50 μg/mL) showed that fluorescent intensity was inversely related to the concentration of protein loaded. These data support 2D FDS as a novel technique in identifying nanoparticles and their surface interactions as a quality assurance tool.

  14. Entangled photon-pair two-dimensional fluorescence spectroscopy (EPP-2DFS).

    PubMed

    Raymer, M G; Marcus, Andrew H; Widom, Julia R; Vitullo, Dashiell L P

    2013-12-12

    We introduce a new method, called entangled photon-pair two-dimensional fluorescence spectroscopy (EPP-2DFS), to sensitively probe the nonlinear electronic response of molecular systems. The method incorporates a separated two-photon ('Franson') interferometer, which generates time-frequency-entangled photon pairs, into the framework of a fluorescence-detected 2D optical spectroscopic experiment. The entangled photons are temporally shaped and phase-modulated in the interferometer, and are used to excite a two-photon-absorbing (TPA) sample, whose excited-state population is selectively detected by simultaneously monitoring the sample fluorescence and the exciting fields. In comparison to 'classical' 2DFS techniques, major advantages of this scheme are the suppression of uncorrelated background signals, the enhancement of simultaneous time-and-frequency resolution, the suppression of diagonal 2D spectral features, and the enhancement and narrowing of off-diagonal spectral cross-peaks that contain information about electronic couplings. These effects are a consequence of the pure-state field properties unique to a parametric down-conversion light source, which must be included in the quantum mechanical description of the composite field-molecule system. We numerically simulate the EPP-2DFS observable for the case of an electronically coupled molecular dimer. The EPP-2DFS spectrum is greatly simplified in comparison to its classical 2D counterpart. Our results indicate that EPP-2DFS can provide previously unattainable resolution to extract model Hamiltonian parameters from electronically coupled molecular dimers.

  15. Two-Dimensional Fluorescence Difference Spectroscopy to Characterize Nanoparticles and their Interactions

    PubMed Central

    Hurst, Miranda N.; DeLong, Robert K.

    2016-01-01

    Two dimensional fluorescence difference spectroscopy (2D FDS) detects nanoparticle interactions following surface functionalization and biomolecule loading by generating a spectral signature of the fluorescent intensity per excitation and emission wavelengths. Comparing metal oxide nanoparticles revealed a unique spectral signature per material composition. 2D FDS showed to be sensitive to changes in surface properties between ZnO NPs synthesized by different methods. ZnO NP loaded with glycol chitosan, polyacrylic acid (PAA), or methoxy polyethylene glycol (mPEG) exhibited a distinct spectral signature shift. ZnO NP loaded with Torula Yeast RNA (TYRNA)(640 nm), polyinosinic: polycytidylic acid (pIC)(680 nm), or splice switching oligonucleotide (SSO)(650 nm) each revealed a shift in emission. Ras-Binding domain (RBD) at three concentrations (25, 37.5, 50 μg/mL) showed that fluorescent intensity was inversely related to the concentration of protein loaded. These data support 2D FDS as a novel technique in identifying nanoparticles and their surface interactions as a quality assurance tool. PMID:27624316

  16. Two-Dimensional Fluorescence Difference Spectroscopy to Characterize Nanoparticles and their Interactions

    NASA Astrophysics Data System (ADS)

    Hurst, Miranda N.; Delong, Robert K.

    2016-09-01

    Two dimensional fluorescence difference spectroscopy (2D FDS) detects nanoparticle interactions following surface functionalization and biomolecule loading by generating a spectral signature of the fluorescent intensity per excitation and emission wavelengths. Comparing metal oxide nanoparticles revealed a unique spectral signature per material composition. 2D FDS showed to be sensitive to changes in surface properties between ZnO NPs synthesized by different methods. ZnO NP loaded with glycol chitosan, polyacrylic acid (PAA), or methoxy polyethylene glycol (mPEG) exhibited a distinct spectral signature shift. ZnO NP loaded with Torula Yeast RNA (TYRNA)(640 nm), polyinosinic: polycytidylic acid (pIC)(680 nm), or splice switching oligonucleotide (SSO)(650 nm) each revealed a shift in emission. Ras-Binding domain (RBD) at three concentrations (25, 37.5, 50 μg/mL) showed that fluorescent intensity was inversely related to the concentration of protein loaded. These data support 2D FDS as a novel technique in identifying nanoparticles and their surface interactions as a quality assurance tool.

  17. Pulse Propagation Effects in Optical 2D Fourier-Transform Spectroscopy: Theory.

    PubMed

    Spencer, Austin P; Li, Hebin; Cundiff, Steven T; Jonas, David M

    2015-04-30

    A solution to Maxwell's equations in the three-dimensional frequency domain is used to calculate rephasing two-dimensional Fourier transform (2DFT) spectra of the D2 line of atomic rubidium vapor in argon buffer gas. Experimental distortions from the spatial propagation of pulses through the sample are simulated in 2DFT spectra calculated for the homogeneous Bloch line shape model. Spectral features that appear at optical densities of up to 3 are investigated. As optical density increases, absorptive and dispersive distortions start with peak shape broadening, progress to peak splitting, and ultimately result in a previously unexplored coherent transient twisting of the split peaks. In contrast to the low optical density limit, where the 2D peak shape for the Bloch model depends only on the total dephasing time, these distortions of the 2D peak shape at finite optical density vary with the waiting time and the excited state lifetime through coherent transient effects. Experiment-specific conditions are explored, demonstrating the effects of varying beam overlap within the sample and of pseudo-time domain filtering. For beam overlap starting at the sample entrance, decreasing the length of beam overlap reduces the line width along the ωτ axis but also reduces signal intensity. A pseudo-time domain filter, where signal prior to the center of the last excitation pulse is excluded from the FID-referenced 2D signal, reduces propagation distortions along the ωt axis. It is demonstrated that 2DFT rephasing spectra cannot take advantage of an excitation-detection transformation that can eliminate propagation distortions in 2DFT relaxation spectra. Finally, the high optical density experimental 2DFT spectrum of rubidium vapor in argon buffer gas [J. Phys. Chem. A 2013, 117, 6279-6287] is quantitatively compared, in line width, in depth of peak splitting, and in coherent transient peak twisting, to a simulation with optical density higher than that reported.

  18. The development of attenuation compensation models of fluorescence spectroscopy signals

    NASA Astrophysics Data System (ADS)

    Dremin, Victor V.; Zherebtsov, Evgeny A.; Rafailov, Ilya E.; Vinokurov, Andrey Y.; Novikova, Irina N.; Zherebtsova, Angelina I.; Litvinova, Karina S.; Dunaev, Andrey V.

    2016-04-01

    This study examines the effect of blood absorption on the endogenous fluorescence signal intensity of biological tissues. Experimental studies were conducted to identify these effects. To register the fluorescence intensity, the fluorescence spectroscopy method was employed. The intensity of the blood flow was measured by laser Doppler flowmetry. We proposed one possible implementation of the Monte Carlo method for the theoretical analysis of the effect of blood on the fluorescence signals. The simulation is constructed as a four-layer skin optical model based on the known optical parameters of the skin with different levels of blood supply. With the help of the simulation, we demonstrate how the level of blood supply can affect the appearance of the fluorescence spectra. In addition, to describe the properties of biological tissue, which may affect the fluorescence spectra, we turned to the method of diffuse reflectance spectroscopy (DRS). Using the spectral data provided by the DRS, the tissue attenuation effect can be extracted and used to correct the fluorescence spectra.

  19. Communication: two-dimensional gas-phase coherent anti-Stokes Raman spectroscopy (2D-CARS): simultaneous planar imaging and multiplex spectroscopy in a single laser shot.

    PubMed

    Bohlin, Alexis; Kliewer, Christopher J

    2013-06-14

    Coherent anti-Stokes Raman spectroscopy (CARS) has been widely used as a powerful tool for chemical sensing, molecular dynamics measurements, and rovibrational spectroscopy since its development over 30 years ago, finding use in fields of study as diverse as combustion diagnostics, cell biology, plasma physics, and the standoff detection of explosives. The capability for acquiring resolved CARS spectra in multiple spatial dimensions within a single laser shot has been a long-standing goal for the study of dynamical processes, but has proven elusive because of both phase-matching and detection considerations. Here, by combining new phase matching and detection schemes with the high efficiency of femtosecond excitation of Raman coherences, we introduce a technique for single-shot two-dimensional (2D) spatial measurements of gas phase CARS spectra. We demonstrate a spectrometer enabling both 2D plane imaging and spectroscopy simultaneously, and present the instantaneous measurement of 15,000 spatially correlated rotational CARS spectra in N2 and air over a 2D field of 40 mm(2).

  20. Communication: Two-dimensional gas-phase coherent anti-Stokes Raman spectroscopy (2D-CARS): Simultaneous planar imaging and multiplex spectroscopy in a single laser shot

    NASA Astrophysics Data System (ADS)

    Bohlin, Alexis; Kliewer, Christopher J.

    2013-06-01

    Coherent anti-Stokes Raman spectroscopy (CARS) has been widely used as a powerful tool for chemical sensing, molecular dynamics measurements, and rovibrational spectroscopy since its development over 30 years ago, finding use in fields of study as diverse as combustion diagnostics, cell biology, plasma physics, and the standoff detection of explosives. The capability for acquiring resolved CARS spectra in multiple spatial dimensions within a single laser shot has been a long-standing goal for the study of dynamical processes, but has proven elusive because of both phase-matching and detection considerations. Here, by combining new phase matching and detection schemes with the high efficiency of femtosecond excitation of Raman coherences, we introduce a technique for single-shot two-dimensional (2D) spatial measurements of gas phase CARS spectra. We demonstrate a spectrometer enabling both 2D plane imaging and spectroscopy simultaneously, and present the instantaneous measurement of 15 000 spatially correlated rotational CARS spectra in N2 and air over a 2D field of 40 mm2.

  1. Communication: Two-dimensional gas-phase coherent anti-Stokes Raman spectroscopy (2D-CARS): Simultaneous planar imaging and multiplex spectroscopy in a single laser shot

    SciTech Connect

    Bohlin, Alexis; Kliewer, Christopher J.

    2013-01-01

    Coherent anti-Stokes Raman spectroscopy (CARS) has been widely used as a powerful tool for chemical sensing, molecular dynamics measurements, and rovibrational spectroscopy since its development over 30 years ago, finding use in fields of study as diverse as combustion diagnostics, cell biology, plasma physics, and the standoff detection of explosives. The capability for acquiring resolved CARS spectra in multiple spatial dimensions within a single laser shot has been a long-standing goal for the study of dynamical processes, but has proven elusive because of both phase-matching and detection considerations. Here, by combining new phase matching and detection schemes with the high efficiency of femtosecond excitation of Raman coherences, we introduce a technique for single-shot two-dimensional (2D) spatial measurements of gas phase CARS spectra. We demonstrate a spectrometer enabling both 2D plane imaging and spectroscopy simultaneously, and present the instantaneous measurement of 15, 000 spatially correlated rotational CARS spectra in N2 and air over a 2D field of 40 mm2.

  2. Analysis of pyruvylated beta-carrageenan by 2D NMR spectroscopy and reductive partial hydrolysis.

    PubMed

    Falshaw, Ruth; Furneaux, Richard H; Wong, Herbert

    2003-06-23

    A polysaccharide rich in 4',6'-O-(1-carboxyethylidene)-substituted (i.e., pyruvylated) beta-carrageenan has been prepared by solvolytic desulfation of a polysaccharide containing predominantly pyruvylated alpha-carrageenan, which was extracted from the red seaweed, Callophycus tridentifer. The 13C and 1H NMR chemical shifts of pyruvylated beta-carrageenan have been fully assigned using 2D NMR spectroscopic techniques. The 4',6'-O-(1-methoxycarbonylethylidene) group, generated during chemical methylation of the polysaccharide, has been shown to survive under the conditions of acidic hydrolysis that cleave the 3,6-anhydro-alpha-D-galactosidic bonds in permethylated samples of both pyruvylated beta- and pyruvylated alpha-carrageenans. As a result, two novel pyruvylated carrabiitol derivatives have been prepared.

  3. "FluSpec": A Simulated Experiment in Fluorescence Spectroscopy

    ERIC Educational Resources Information Center

    Bigger, Stephen W.; Bigger, Andrew S.; Ghiggino, Kenneth P.

    2014-01-01

    The "FluSpec" educational software package is a fully contained tutorial on the technique of fluorescence spectroscopy as well as a simulator on which experiments can be performed. The procedure for each of the experiments is also contained within the package along with example analyses of results that are obtained using the software.

  4. Preparation and characterization of CdSe colloidal quantum dots by pptical spectroscopy and 2D DOSY NMR

    NASA Astrophysics Data System (ADS)

    Geru, I.; Bordian, O.; Culeac, I.; Turta, C.; Verlan, V.; Barba, A.

    2015-02-01

    We present experimental results on preparation and characterization of colloidal CdSe quantum dots (QD) in organic solvent. CdSe QDs were synthesized following a modified literature method and have been characterized by UV-Vis absorption and photoluminescent (PL) spectroscopy, as well as by 2D Diffusion Ordered Spectroscopy (DOSY) NMR. The average CdSe particles size estimated from the UV-Vis absorption spectra was found to be in the range 2.28 - 2.92 nm, which correlates very well with the results obtained from NMR measurements. The PL spectrum for CdSe nanodots can be characterized by a narrow emission band with the peak maximum shifting from 508 to 566 nm in dependence of the CdSe nanoparticle size. The PL is dominated by a near-band-edge emission, accompanied by a weak broad band in the near IR, related to the surface shallow trap emission.

  5. Deconvolution of 2D coincident Doppler broadening spectroscopy using the Richardson Lucy algorithm

    NASA Astrophysics Data System (ADS)

    Zhang, J. D.; Zhou, T. J.; Cheung, C. K.; Beling, C. D.; Fung, S.; Ng, M. K.

    2006-05-01

    Coincident Doppler Broadening Spectroscopy (CDBS) measurements are popular in positron solid-state studies of materials. By utilizing the instrumental resolution function obtained from a gamma line close in energy to the 511 keV annihilation line, it is possible to significantly enhance the quality of the CDBS spectra using deconvolution algorithms. In this paper, we compare two algorithms, namely the Non-Negativity Least Squares (NNLS) regularized method and the Richardson-Lucy (RL) algorithm. The latter, which is based on the method of maximum likelihood, is found to give superior results to the regularized least-squares algorithm and with significantly less computer processing time.

  6. 2D Raman spectroscopy as an alternative technique for distinguishing oleanoic acid and ursolic acid

    NASA Astrophysics Data System (ADS)

    Mello, César; Crotti, Antônio E. M.; Vessecchi, Ricardo; Cunha, Wilson R.

    2006-11-01

    The isomeric triterpenes oleanoic acid and ursolic acid are compounds exhibiting a variety of biological activities. Structurally, they differ only in the position of the methyl group (C-29) at ring E. The differentiation of these two compounds requires a detailed analysis of their 13C and 1H NMR spectra which is often tedious and time-consuming, besides the need of using deuterated solvents. In this work, we report the use of bidimensional Raman spectroscopy as a fast technique to distinguish these two bioactive isomeric compounds.

  7. Combined fiber probe for fluorescence lifetime and Raman spectroscopy.

    PubMed

    Dochow, Sebastian; Ma, Dinglong; Latka, Ines; Bocklitz, Thomas; Hartl, Brad; Bec, Julien; Fatakdawala, Hussain; Marple, Eric; Urmey, Kirk; Wachsmann-Hogiu, Sebastian; Schmitt, Michael; Marcu, Laura; Popp, Jürgen

    2015-11-01

    In this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. Graphical Abstract An image comparison between FLIm and Raman spectroscopy acquired with the bimodal probe onseveral tissue samples.

  8. Chemical profiling and adulteration screening of Aquilariae Lignum Resinatum by Fourier transform infrared (FT-IR) spectroscopy and two-dimensional correlation infrared (2D-IR) spectroscopy.

    PubMed

    Qu, Lei; Chen, Jian-Bo; Zhang, Gui-Jun; Sun, Su-Qin; Zheng, Jing

    2017-03-05

    As a kind of expensive perfume and valuable herb, Aquilariae Lignum Resinatum (ALR) is often adulterated for economic motivations. In this research, Fourier transform infrared (FT-IR) spectroscopy is employed to establish a simple and quick method for the adulteration screening of ALR. First, the principal chemical constituents of ALR are characterized by FT-IR spectroscopy at room temperature and two-dimensional correlation infrared (2D-IR) spectroscopy with thermal perturbation. Besides the common cellulose and lignin compounds, a certain amount of resin is the characteristic constituent of ALR. Synchronous and asynchronous 2D-IR spectra indicate that the resin (an unstable secondary metabolite) is more sensitive than cellulose and lignin (stable structural constituents) to the thermal perturbation. Using a certified ALR sample as the reference, the infrared spectral correlation threshold is determined by 30 authentic samples and 6 adulterated samples. The spectral correlation coefficient of an authentic ALR sample to the standard reference should be not less than 0.9886 (p=0.01). Three commercial adulterated ALR samples are identified by the correlation threshold. Further interpretation of the infrared spectra of the adulterated samples indicates the common adulterating methods - counterfeiting with other kind of wood, adding ingredient such as sand to increase the weight, and adding the cheap resin such as rosin to increase the content of resin compounds. Results of this research prove that FT-IR spectroscopy can be used as a simple and accurate quality control method of ALR.

  9. Chemical profiling and adulteration screening of Aquilariae Lignum Resinatum by Fourier transform infrared (FT-IR) spectroscopy and two-dimensional correlation infrared (2D-IR) spectroscopy

    NASA Astrophysics Data System (ADS)

    Qu, Lei; Chen, Jian-bo; Zhang, Gui-Jun; Sun, Su-qin; Zheng, Jing

    2017-03-01

    As a kind of expensive perfume and valuable herb, Aquilariae Lignum Resinatum (ALR) is often adulterated for economic motivations. In this research, Fourier transform infrared (FT-IR) spectroscopy is employed to establish a simple and quick method for the adulteration screening of ALR. First, the principal chemical constituents of ALR are characterized by FT-IR spectroscopy at room temperature and two-dimensional correlation infrared (2D-IR) spectroscopy with thermal perturbation. Besides the common cellulose and lignin compounds, a certain amount of resin is the characteristic constituent of ALR. Synchronous and asynchronous 2D-IR spectra indicate that the resin (an unstable secondary metabolite) is more sensitive than cellulose and lignin (stable structural constituents) to the thermal perturbation. Using a certified ALR sample as the reference, the infrared spectral correlation threshold is determined by 30 authentic samples and 6 adulterated samples. The spectral correlation coefficient of an authentic ALR sample to the standard reference should be not less than 0.9886 (p = 0.01). Three commercial adulterated ALR samples are identified by the correlation threshold. Further interpretation of the infrared spectra of the adulterated samples indicates the common adulterating methods - counterfeiting with other kind of wood, adding ingredient such as sand to increase the weight, and adding the cheap resin such as rosin to increase the content of resin compounds. Results of this research prove that FT-IR spectroscopy can be used as a simple and accurate quality control method of ALR.

  10. Ionic Liquid–Solute Interactions Studied by 2D NOE NMR Spectroscopy

    SciTech Connect

    Khatun, Sufia; Castner, Edward W.

    2014-11-26

    Intermolecular interactions between a Ru²⁺(bpy)₃ solute and the anions and cations of four different ionic liquids (ILs) are investigated by 2D NMR nuclear Overhauser effect (NOE) techniques, including {¹H-¹⁹F} HOESY and {¹H-¹H} ROESY. Four ILs are studied, each having the same bis(trifluoromethylsulfonyl)amide anion in common. Two of the ILs have aliphatic 1-alkyl-1-methylpyrrolidinium cations, while the other two ILs have aromatic 1-alkyl-3-methylimidazolium cations. ILs with both shorter (butyl) and longer (octyl or decyl) cationic alkyl substituents are studied. NOE NMR results suggest that the local environment of IL anions and cations near the Ru²⁺(bpy)₃ solute is rather different from the bulk IL structure. The solute-anion and solute-cation interactions are significantly different both for ILs with short vs long alkyl tails and for ILs with aliphatic vs aromatic cation polar head groups. In particular, the solute-anion interactions are observed to be about 3 times stronger for the cations with shorter alkyl tails relative to the ILs with longer alkyl tails. The Ru²⁺(bpy)₃ solute interacts with both the polar head and the nonpolar tail groups of the 1- butyl-1-methylpyrrolidinium cation but only with the nonpolar tail groups of the 1-decyl-1-methylpyrrolidinium cation.

  11. Ionic Liquid–Solute Interactions Studied by 2D NOE NMR Spectroscopy

    DOE PAGES

    Khatun, Sufia; Castner, Edward W.

    2014-11-26

    Intermolecular interactions between a Ru²⁺(bpy)₃ solute and the anions and cations of four different ionic liquids (ILs) are investigated by 2D NMR nuclear Overhauser effect (NOE) techniques, including {¹H-¹⁹F} HOESY and {¹H-¹H} ROESY. Four ILs are studied, each having the same bis(trifluoromethylsulfonyl)amide anion in common. Two of the ILs have aliphatic 1-alkyl-1-methylpyrrolidinium cations, while the other two ILs have aromatic 1-alkyl-3-methylimidazolium cations. ILs with both shorter (butyl) and longer (octyl or decyl) cationic alkyl substituents are studied. NOE NMR results suggest that the local environment of IL anions and cations near the Ru²⁺(bpy)₃ solute is rather different from the bulkmore » IL structure. The solute-anion and solute-cation interactions are significantly different both for ILs with short vs long alkyl tails and for ILs with aliphatic vs aromatic cation polar head groups. In particular, the solute-anion interactions are observed to be about 3 times stronger for the cations with shorter alkyl tails relative to the ILs with longer alkyl tails. The Ru²⁺(bpy)₃ solute interacts with both the polar head and the nonpolar tail groups of the 1- butyl-1-methylpyrrolidinium cation but only with the nonpolar tail groups of the 1-decyl-1-methylpyrrolidinium cation.« less

  12. Sucrose Monoester Micelles Size Determined by Fluorescence Correlation Spectroscopy (FCS)

    PubMed Central

    Sanchez, Susana A.; Gratton, Enrico; Zanocco, Antonio L.; Lemp, Else; Gunther, German

    2011-01-01

    One of the several uses of sucrose detergents, as well as other micelle forming detergents, is the solubilization of different membrane proteins. Accurate knowledge of the micelle properties, including size and shape, are needed to optimize the surfactant conditions for protein purification and membrane characterization. We synthesized sucrose esters having different numbers of methylene subunits on the substituent to correlate the number of methylene groups with the size of the corresponding micelles. We used Fluorescence Correlation Spectroscopy (FCS) and two photon excitation to determine the translational D of the micelles and calculate their corresponding hydrodynamic radius, Rh. As a fluorescent probe we used LAURDAN (6-dodecanoyl-2-dimethylaminonaphthalene), a dye highly fluorescent when integrated in the micelle and non-fluorescent in aqueous media. We found a linear correlation between the size of the tail and the hydrodynamic radius of the micelle for the series of detergents measured. PMID:22216230

  13. Single-molecule fluorescence spectroscopy in (bio)catalysis.

    PubMed

    Roeffaers, Maarten B J; De Cremer, Gert; Uji-i, Hiroshi; Muls, Benîot; Sels, Bert F; Jacobs, Pierre A; De Schryver, Frans C; De Vos, Dirk E; Hofkens, Johan

    2007-07-31

    The ever-improving time and space resolution and molecular detection sensitivity of fluorescence microscopy offer unique opportunities to deepen our insights into the function of chemical and biological catalysts. Because single-molecule microscopy allows for counting the turnover events one by one, one can map the distribution of the catalytic activities of different sites in solid heterogeneous catalysts, or one can study time-dependent activity fluctuations of individual sites in enzymes or chemical catalysts. By experimentally monitoring individuals rather than populations, the origin of complex behavior, e.g., in kinetics or in deactivation processes, can be successfully elucidated. Recent progress of temporal and spatial resolution in single-molecule fluorescence microscopy is discussed in light of its impact on catalytic assays. Key concepts are illustrated regarding the use of fluorescent reporters in catalytic reactions. Future challenges comprising the integration of other techniques, such as diffraction, scanning probe, or vibrational methods in single-molecule fluorescence spectroscopy are suggested.

  14. Laser Induced Fluorescence Spectroscopy of Cobalt Monoboride

    NASA Astrophysics Data System (ADS)

    Pang, H. F.; Ng, Y. W.; Cheung, A. S.-C.

    2011-06-01

    Laser induced fluorescence spectrum of cobalt monoboride (CoB) in the visible region between 465 and 560 nm has been observed. CoB molecule was produced by the reaction of laser ablated cobalt atom and diborane (B_2H_6) seeded in argon. Over twenty five vibronic bands have been recorded, and both Co10B and Co11B isotopic species have been observed and analyzed. Preliminary analysis of the rotational lines showed that the observed vibronic bands belong to two categories namely: the Ω' = 2 - Ω'' = 2 and the Ω' = 3 - Ω'' = 3 transitions, which indicated the ground state of CoB is consistent with an assignment of a ^3Δ_i state predicted from ab initio calculations. Unresolved hyperfine structure arising from the Co nucleus (I = 7/2) causes a broadening of spectral lines. This work represents the first experimental investigation of the spectrum of the CoB molecule. Financial support from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. HKU 701008P) is gratefully acknowledged.

  15. Fluorescence spectroscopy to assess apoptosis in myocardium

    NASA Astrophysics Data System (ADS)

    Ranji, Mahsa; Matsubara, Muneaki; Grosso, Michael A.; Jaggard, Dwight L.; Chance, Britton; Gorman, Robert C.; Gorman, Joseph H., III

    2007-02-01

    Apoptosis induced mitochondrial destruction and dysfunction has been shown to play an important role in the pathogenesis of both acute cardiac ischemia-reperfusion injury and chronic myocardial infarction-induced ventricular remodeling. Unfortunately this understanding has not translated into effective therapeutic strategies for either condition-mostly due to an inability to assess mitochondrial dysfunction/apoptosis effectively in humans. All current measures of apoptosis are pseudo-quantitative and require invasive tissue biopsy. Our group has developed an optical, non-tissue destructive catheter based device that allows the quantitative regional assessment of this pathological process in vivo. This instrument has been designed to acquire fluorescence signals of intrinsic mitochondrial fluorophores, Nicotinamide Adenine Dinucleotide (NAD) and Flavoprotein (FP). The normalized ratio of these fluorophores (FP/FP+NADH) called the redox ratio, is an indicator of the in vivo mitochondrial dysfunction. 1-3 We have demonstrated in a rabbit reperfusion model of apoptotic myocyte injury that this redox ratio is drastically increased which is consistent with profound apoptosis-induced "unhinging" of the mitochondrial respiratory function.

  16. Beyond the Born-Oppenheimer approximation: High-resolution overtone spectroscopy of H2D+ and D2H+

    NASA Astrophysics Data System (ADS)

    Fárník, Michal; Davis, Scott; Kostin, Maxim A.; Polyansky, Oleg L.; Tennyson, Jonathan; Nesbitt, David J.

    2002-04-01

    Transitions to overtone 2ν2 and 2ν3, and combination ν2+ν3 vibrations in jet-cooled H2D+ and D2H+ molecular ions have been measured for the first time by high-resolution IR spectroscopy. The source of these ions is a pulsed slit jet supersonic discharge, which allows for efficient generation, rotational cooling, and high frequency (100 KHz) concentration modulation for detection via sensitive lock-in detection methods. Isotopic substitution and high-resolution overtone spectroscopy in this fundamental molecular ion permit a systematic, first principles investigation of Born-Oppenheimer "breakdown" effects due to large amplitude vibrational motion as well as provide rigorous tests of approximate theoretical methods beyond the Born-Oppenheimer level. The observed overtone transitions are in remarkably good agreement (<0.1 cm-1) with non-Born-Oppenheimer ab initio theoretical predictions, with small but systematic deviations for 2ν2, ν2+ν3, and 2ν3 excited states indicating directions for further improvement in such treatments. Spectroscopic assignment and analysis of the isotopomeric transitions reveals strong Coriolis mixing between near resonant 2ν3 and ν2+ν3 vibrations in D2H+. Population-independent line intensity ratios for transitions from common lower states indicate excellent overall agreement with theoretical predictions for D2H+, but with statistically significant discrepancies noted for H2D+. Finally, H2D+ versus D2H+ isotopomer populations are analyzed as a function of D2/H2 mixing ratio and can be well described by steady state kinetics in the slit discharge expansion.

  17. Rapid identification of Pterocarpus santalinus and Dalbergia louvelii by FTIR and 2D correlation IR spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Fang-Da; Xu, Chang-Hua; Li, Ming-Yu; Huang, An-Min; Sun, Su-Qin

    2014-07-01

    Since Pterocarpus santalinus and Dalbergia louvelii, which are of precious Rosewood, are very similar in their appearance and anatomy characteristics, cheaper Hongmu D. louvelii is often illegally used to impersonate valuable P. santalinus, especially in Chinese furniture manufacture. In order to develop a rapid and effective method for easy confused wood furniture differentiation, we applied tri-step identification method, i.e., conventional infrared spectroscopy (FT-IR), second derivative infrared (SD-IR) spectroscopy and two-dimensional correlation infrared (2DCOS-IR) spectroscopy to investigate P. santalinus and D. louvelii furniture. According to FT-IR and SD-IR spectra, it has been found two unconditional stable difference at 848 cm-1 and 700 cm-1 and relative stable differences at 1735 cm-1, 1623 cm-1, 1614 cm-1, 1602 cm-1, 1509 cm-1, 1456 cm-1, 1200 cm-1, 1158 cm-1, 1055 cm-1, 1034 cm-1 and 895 cm-1 between D. louvelii and P. santalinus IR spectra. The stable discrepancy indicates that the category of extractives is different between the two species. Besides, the relative stable differences imply that the content of holocellulose in P. santalinus is more than that of D. louvelii, whereas the quantity of extractives in D. louvelii is higher. Furthermore, evident differences have been observed in their 2DCOS-IR spectra of 1550-1415 cm-1 and 1325-1030 cm-1. P. santalinus has two strong auto-peaks at 1459 cm-1 and 1467 cm-1, three mid-strong auto-peaks at 1518 cm-1, 1089 cm-1 and 1100 cm-1 and five weak auto-peaks at 1432 cm-1, 1437 cm-1, 1046 cm-1, 1056 cm-1 and 1307 cm-1 while D. louvelii has four strong auto-peaks at 1465 cm-1, 1523 cm-1, 1084 cm-1 and 1100 cm-1, four mid-strong auto-peaks at 1430 cm-1, 1499 cm-1, 1505 cm-1 and 1056 cm-1 and two auto-peaks at 1540 cm-1 and 1284 cm-1. This study has proved that FT-IR integrated with 2DCOS-IR could be applicable for precious wood furniture authentication in a direct, rapid and holistic manner.

  18. Performance improvements in temperature reconstructions of 2-D tunable diode laser absorption spectroscopy (TDLAS)

    NASA Astrophysics Data System (ADS)

    Choi, Doo-Won; Jeon, Min-Gyu; Cho, Gyeong-Rae; Kamimoto, Takahiro; Deguchi, Yoshihiro; Doh, Deog-Hee

    2016-02-01

    Performance improvement was attained in data reconstructions of 2-dimensional tunable diode laser absorption spectroscopy (TDLAS). Multiplicative Algebraic Reconstruction Technique (MART) algorithm was adopted for data reconstruction. The data obtained in an experiment for the measurement of temperature and concentration fields of gas flows were used. The measurement theory is based upon the Beer-Lambert law, and the measurement system consists of a tunable laser, collimators, detectors, and an analyzer. Methane was used as a fuel for combustion with air in the Bunsen-type burner. The data used for the reconstruction are from the optical signals of 8-laser beams passed on a cross-section of the methane flame. The performances of MART algorithm in data reconstruction were validated and compared with those obtained by Algebraic Reconstruction Technique (ART) algorithm.

  19. Characterization of metabolites in different kiwifruit varieties by NMR and fluorescence spectroscopy.

    PubMed

    Abdul Hamid, Nur Ashikin; Mediani, Ahmed; Maulidiani, M; Abas, Faridah; Park, Yong Seo; Leontowicz, Hanna; Leontowicz, Maria; Namiesnik, Jacek; Gorinstein, S

    2017-05-10

    It is known from our previous studies that kiwifruits, which are used in common human diet, have preventive properties of coronary artery disease. This study describes a combination of (1)H NMR spectroscopy, multivariate data analyses and fluorescence measurements in differentiating of some kiwifruit varieties, their quenching and antioxidant properties. A total of 41 metabolites were identified by comparing with literature data Chenomx database and 2D NMR. The binding properties of the extracted polyphenols against HSA showed higher reactivity of studied two cultivars in comparison with the common Hayward. The results showed that the fluorescence of HSA was quenched by Bidan as much as twice than by other fruits. The correlation between the binding properties of polyphenols in the investigated fruits, their relative quantification and suggested metabolic pathway was established. These results can provide possible application of fruit extracts in pharmaceutical industry.

  20. Positron spectroscopy of 2D materials using an advanced high intensity positron beam

    NASA Astrophysics Data System (ADS)

    McDonald, A.; Chirayath, V.; Lim, Z.; Gladen, R.; Chrysler, M.; Fairchild, A.; Koymen, A.; Weiss, A.

    An advanced high intensity variable energy positron beam(~1eV to 20keV) has been designed, tested and utilized for the first coincidence Doppler broadening (CDB) measurements on 6-8 layers graphene on polycrystalline Cu sample. The system is capable of simultaneous Positron annihilation induced Auger electron Spectroscopy (PAES) and CDB measurements giving it unparalleled sensitivity to chemical structure at external surfaces, interfaces and internal pore surfaces. The system has a 3m flight path up to a micro channel plate (MCP) for the Auger electrons emitted from the sample. This gives a superior energy resolution for PAES. A solid rare gas(Neon) moderator was used for the generation of the monoenergetic positron beam. The positrons were successfully transported to the sample chamber using axial magnetic field generated with a series of Helmholtz coils. We will discuss the PAES and coincidence Doppler broadening measurements on graphene -Cu sample and present an analysis of the gamma spectra which indicates that a fraction of the positrons implanted at energies 7-60eV can become trapped at the graphene/metal interface. This work was supported by NSF Grant No. DMR 1508719 and DMR 1338130.

  1. Femtosecond broadband fluorescence upconversion spectroscopy: Improved setup and photometric correction

    SciTech Connect

    Zhang, X.-X.; Wuerth, C.; Resch-Genger, U.; Zhao, L.; Ernsting, N. P.; Sajadi, M.

    2011-06-15

    A setup for fluorescence upconversion spectroscopy (FLUPS) is described which has 80 fs temporal response (fwhm) for emission in the spectral range 425-750 nm. Broadband phase matching is achieved with tilted gate pulses at 1340 nm. Background from harmonics of the gate pulse is removed and sensitivity increased compared to previous designs. Photometric calibration of the upconversion process is performed with a set of fluorescent dyes. For Coumarin 153 in methanol the peak position, bandwidth, and asymmetry depending on delay time are reported.

  2. Diffusivity of asphaltene molecules by fluorescence correlation spectroscopy.

    PubMed

    Andrews, A Ballard; Guerra, Rodrigo E; Mullins, Oliver C; Sen, Pabitra N

    2006-07-06

    Using fluorescence correlation spectroscopy (FCS) we measure the translational diffusion coefficient of asphaltene molecules in toluene at extremely low concentrations (0.03-3.0 mg/L): where aggregation does not occur. We find that the translational diffusion coefficient of asphaltene molecules in toluene is about 0.35 x 10(-5) cm(2)/s at room temperature. This diffusion coefficient corresponds to a hydrodynamic radius of approximately 1 nm. These data confirm previously estimated size from rotational diffusion studied using fluorescence depolarization. The implication of this concurrence is that asphaltene molecular structures are monomeric, not polymeric.

  3. Fluorescence correlation spectroscopy: Diagnostics for sparse molecules

    PubMed Central

    Maiti, Sudipta; Haupts, Ulrich; Webb, Watt W.

    1997-01-01

    The robust glow of molecular fluorescence renders even sparse molecules detectable and susceptible to analysis for concentration, mobility, chemistry, and photophysics. Correlation spectroscopy, a statistical-physics-based tool, gleans quantitative information from the spontaneously fluctuating fluorescence signals obtained from small molecular ensembles. This analytical power is available for studying molecules present at minuscule concentrations in liquid solutions (less than one nanomolar), or even on the surfaces of living cells at less than one macromolecule per square micrometer. Indeed, routines are becoming common to detect, locate, and examine individual molecules under favorable conditions. PMID:9342306

  4. Optical-fiber-microsphere for remote fluorescence correlation spectroscopy.

    PubMed

    Aouani, Heykel; Deiss, Frédérique; Wenger, Jérôme; Ferrand, Patrick; Sojic, Neso; Rigneault, Hervé

    2009-10-12

    Fluorescence correlation spectroscopy (FCS) is a versatile method that would greatly benefit to remote optical-fiber fluorescence sensors. However, the current state-of-the-art struggles with high background and low detection sensitivities that prevent the extension of fiber-based FCS down to the single-molecule level. Here we report the use of an optical fiber combined with a latex microsphere to perform FCS analysis. The sensitivity of the technique is demonstrated at the single molecule level thanks to a photonic nanojet effect. This offers new opportunities for reducing the bulky microscope setup and extending FCS to remote or in vivo applications.

  5. Fluorescence suppression using micro-scale spatially offset Raman spectroscopy.

    PubMed

    Conti, Claudia; Botteon, Alessandra; Colombo, Chiara; Realini, Marco; Matousek, Pavel

    2016-09-21

    We present a new concept of fluorescence suppression in Raman microscopy based on micro-spatially offset Raman spectroscopy which is applicable to thin stratified turbid (diffusely scattering) matrices permitting the retrieval of the Raman signals of sublayers below intensely fluorescing turbid over-layers. The method is demonstrated to yield good quality Raman spectra with dramatically suppressed fluorescence backgrounds enabling the retrieval of Raman sublayer signals even in situations where conventional Raman microscopy spectra are fully overwhelmed by intense fluorescence. The concept performance was studied theoretically using Monte Carlo simulations indicating the potential of up to an order or two of magnitude suppression of overlayer fluorescence backgrounds relative to the Raman sublayer signals. The technique applicability was conceptually demonstrated on layered samples involving paints, polymers and stones yielding fluorescence suppression factors between 12 to above 430. The technique has potential applications in a number of analytical areas including cultural heritage, archaeology, polymers, food, pharmaceutical, biological, biomedical, forensics and catalytic sciences and quality control in manufacture.

  6. Fluorescence spectroscopy of excitation transfer in Photosystem 1

    SciTech Connect

    Mukerji, I.

    1990-12-01

    This thesis centers on the study of excitation transfer in a photosynthetic antenna array. The spectroscopic properties of two pigment-protein complexes were investigated. These complexes, isolated from higher plants, display an unusual temperature dependent fluorescence behavior. The author have chosen to study this fluorescence behavior with respect to energy transfer to the reaction center and in an isolated intact antenna preparation. A Photosystem 1 complex, PSI-200, was isolated from spinach. We have characterized this system by both steady state and time-resolved fluorescence spectroscopy. Fluorescence polarization measurements indicate that this emission arises from pigments which absorb in the long wavelength region of the spectrum and comprise a relatively small portion of the antenna population. Comparison of spectral characteristics were made with a PSI complex isolated from the thermophilic cyanobacterium, Synechococcus, sp. To address the role of Chl b in stimulating long wavelength fluorescence and the temperature dependence of the system, we have studied the energy transfer dynamics in an antenna complex, LHC-I isolated from PSI-200. Kinetic measurements indicate that initially absorbed excitation is rapidly redistributed to longer wavelength emitting pigments within 40 ps. The temperature dependence of F685 results from increased back transfer from long wavelength emitters to F685. We suggest that changes in excitation transfer between the various emitting species and a non-radiative fluorescence quenching mechanism account for the temperature dependence of the system. 144 refs., 50 figs., 3 tabs.

  7. Investigation on the interaction of cefpirome sulfate with lysozyme by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy.

    PubMed

    Han, Rong; Liu, Baosheng; Li, Gaixia; Zhang, Qiuju

    2016-03-01

    The reaction mechanism of cefpirome sulfate with lysozyme at different temperatures (298, 310 and 318 K) was investigated using fluorescence quenching and synchronous fluorescence spectroscopy under simulated physiological conditions. The results clearly demonstrated that cefpirome sulfate caused strong quenching of the fluorescence of lysozyme by a static quenching mechanism. The binding constants obtained using the above methods were of the same order of magnitude and very similar. Static electric forces played a key role in the interaction between cefpirome sulfate and lysozyme, and the number of binding sites in the interaction was close to 1. The values of Hill's coefficients were > 1, indicating that drugs or proteins showed a very weakly positive cooperativity in the system. In addition, the conclusions obtained from the two methods using the same equation were consistent. The results indicated that synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the fluorescence quenching method. In addition, the effect of cefpirome sulfate on the secondary structure of lysozyme was analyzed using circular dichroism spectroscopy.

  8. Emerging applications of fluorescence spectroscopy in medical microbiology field.

    PubMed

    Shahzad, Aamir; Köhler, Gottfried; Knapp, Martin; Gaubitzer, Erwin; Puchinger, Martin; Edetsberger, Michael

    2009-11-26

    There are many diagnostic techniques and methods available for diagnosis of medically important microorganisms like bacteria, viruses, fungi and parasites. But, almost all these techniques and methods have some limitations or inconvenience. Most of these techniques are laborious, time consuming and with chances of false positive or false negative results. It warrants the need of a diagnostic technique which can overcome these limitations and problems. At present, there is emerging trend to use Fluorescence spectroscopy as a diagnostic as well as research tool in many fields of medical sciences. Here, we will critically discuss research studies which propose that Fluorescence spectroscopy may be an excellent diagnostic as well as excellent research tool in medical microbiology field with high sensitivity and specificity.

  9. Emerging applications of fluorescence spectroscopy in medical microbiology field

    PubMed Central

    2009-01-01

    There are many diagnostic techniques and methods available for diagnosis of medically important microorganisms like bacteria, viruses, fungi and parasites. But, almost all these techniques and methods have some limitations or inconvenience. Most of these techniques are laborious, time consuming and with chances of false positive or false negative results. It warrants the need of a diagnostic technique which can overcome these limitations and problems. At present, there is emerging trend to use Fluorescence spectroscopy as a diagnostic as well as research tool in many fields of medical sciences. Here, we will critically discuss research studies which propose that Fluorescence spectroscopy may be an excellent diagnostic as well as excellent research tool in medical microbiology field with high sensitivity and specificity. PMID:19941643

  10. Near-infrared (NIR) monitoring of Nylon 6 during quenching studied by projection two-dimensional (2D) correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Shinzawa, Hideyuki; Mizukado, Junji

    2016-11-01

    Evolutionary change in supermolecular structure of Nylon 6 during its melt-quenched process was studied by Near-infrared (NIR) spectroscopy. Time-resolved NIR spectra was measured by taking the advantage of high-speed NIR monitoring based on an acousto-optic tunable filter (AOTF). Fine spectral features associated with the variation of crystalline and amorphous structure occurring in relatively short time scale were readily captured. For example, synchronous and asynchronous 2D correlation spectra reveal the initial decrease in the contribution of the NIR band at 1485 nm due to the amorphous structure, predominantly existing in the melt Nylon 6. This is then followed by the emerging contribution of the band intensity at 1535 nm associated with the crystalline structure. Consequently, the results clearly demonstrate a definite advantage of the high-speed NIR monitoring for analyzing fleeting phenomena.

  11. Rotational spectroscopy of vibrationally excited N2H+ and N2D+ up to 2.7 THz

    NASA Astrophysics Data System (ADS)

    Yu, S.; Pearson, J. C.; Drouin, B. J.; Crawford, T.; Daly, A. M.; Elliott, B.; Amano, T.

    2015-08-01

    Terahertz absorption spectroscopy was employed to extend the measurements on the pure rotational transitions of N2H+, N2D+ and their 15N-containing isotopologues in the ground state and first excited vibrational states for the three fundamental vibrational modes. In total, 91 new pure rotational transitions were observed in the range of 0.7-2.7 THz. The observed transition frequencies were fit to experimental accuracy, and the improved molecular parameters were obtained. The new measurements and predictions reported here will support the analysis of high-resolution astronomical observations made with facilities such as SOFIA and ALMA where laboratory rest frequencies with uncertainties of 1 MHz or smaller are required for proper analysis of velocity resolved astrophysical components.

  12. Two-dimensional (2D) Chemiluminescence (CL) correlation spectroscopy for studying thermal oxidation of isotactic polypropylene (iPP)

    NASA Astrophysics Data System (ADS)

    Shinzawa, Hideyuki; Hagihara, Hideaki; Suda, Hiroyuki; Mizukado, Jyunji

    2016-11-01

    Application of the two-dimensional (2D) correlation spectroscopy is extended to Chemiluminescence (CL) spectra of isotactic polypropylene (iPP) under thermally induced oxidation. Upon heating, the polymer chains of the iPP undergoes scissoring and fragmentation to develop several intermediates. While different chemical species provides the emission at different wavelength regions, entire feature of the time-dependent CL spectra of the iPP samples were complicated by the presence of overlapped contributions from singlet oxygen (1O2) and carbonyl species within sample. 2D correlation spectra showed notable enhancement of the spectral resolution to provide penetrating insight into the thermodynamics of the polymer system. For example, the, oxidation induce scissoring and fragmentation of the polymer chains to develop the carbonyl group. Further reaction results in the consumption of the carbonyl species and subsequent production of different 1O2 species each developed in different manner. Consequently, key information on the thermal oxidation can be extracted in a surprisingly simple manner without any analytical expression for the actual response curves of spectral intensity signals during the reaction.

  13. Solving structure in the CP29 light harvesting complex with polarization-phased 2D electronic spectroscopy

    PubMed Central

    Ginsberg, Naomi S.; Davis, Jeffrey A.; Ballottari, Matteo; Cheng, Yuan-Chung; Bassi, Roberto; Fleming, Graham R.

    2011-01-01

    The CP29 light harvesting complex from green plants is a pigment-protein complex believed to collect, conduct, and quench electronic excitation energy in photosynthesis. We have spectroscopically determined the relative angle between electronic transition dipole moments of its chlorophyll excitation energy transfer pairs in their local protein environments without relying on simulations or an X-ray crystal structure. To do so, we measure a basis set of polarized 2D electronic spectra and isolate their absorptive components on account of the tensor relation between the light polarization sequences used to obtain them. This broadly applicable advance further enhances the acuity of polarized 2D electronic spectroscopy and provides a general means to initiate or feed back on the structural modeling of electronically-coupled chromophores in condensed phase systems, tightening the inferred relations between the spatial and electronic landscapes of ultrafast energy flow. We also discuss the pigment composition of CP29 in the context of light harvesting, energy channeling, and photoprotection within photosystem II. PMID:21321222

  14. 2D IR spectroscopy of histidine: probing side-chain structure and dynamics via backbone amide vibrations.

    PubMed

    Ghosh, Ayanjeet; Tucker, Matthew J; Gai, Feng

    2014-07-17

    It is well known that histidine is involved in many biological functions due to the structural versatility of its side chain. However, probing the conformational transitions of histidine in proteins, especially those occurring on an ultrafast time scale, is difficult. Herein we show, using a histidine dipeptide as a model, that it is possible to probe the tautomer and protonation status of a histidine residue by measuring the two-dimensional infrared (2D IR) spectrum of its amide I vibrational transition. Specifically, for the histidine dipeptide studied, the amide unit of the histidine gives rise to three spectrally resolvable amide I features at approximately 1630, 1644, and 1656 cm(-1), respectively, which, based on measurements at different pH values and frequency calculations, are assigned to a τ tautomer (1630 cm(-1) component) and a π tautomer with a hydrated (1644 cm(-1) component) or dehydrated (1656 cm(-1) component) amide. Because of the intrinsic ultrafast time resolution of 2D IR spectroscopy, we believe that the current approach, when combined with the isotope editing techniques, will be useful in revealing the structural dynamics of key histidine residues in proteins that are important for function.

  15. Simultaneous Surface-Near and Solution Fluorescence Correlation Spectroscopy.

    PubMed

    Winterflood, Christian M; Seeger, Stefan

    2016-05-01

    We report the first simultaneous measurement of surface-confined and solution fluorescence correlation spectroscopy (FCS). We use an optical configuration for tightly focused excitation and separate detection of light emitted below (undercritical angle fluorescence, UAF) and above (supercritical angle fluorescence, SAF) the critical angle of total internal reflection of the coverslip/sample interface. This creates two laterally coincident detection volumes which differ in their axial extent. While detection of far-field UAF emission producesa standard confocal volume, near-field-mediated SAF produces a highly surface-confined detection volume at the coverslip/sample interface which extends only ~200 nm into the sample. A characterization of the two detection volumes by FCS of free diffusion is presented and compared with analytical models and simulations. The presented FCS technique allows to determine bulk solution concentrations and surface-near concentrations at the same time.

  16. Principles and applications of fluorescence lifetime correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Beranová, Lenka; Humpolícková, Jana; Hof, Martin

    2009-05-01

    Two fluorescence spectroscopy concepts, fluorescence correlation spectroscopy and time correlated single photon counting (TCSPC) are employed in fluorescence lifetime correlation spectroscopy (FLCS) - a relatively new technique with several experimental benefits. In FLCS experiments, pulsed excitation is used and data are stored in a special time-tagged time-resolved mode. Mathematical treatment of TCSPC decay patterns of distinct fluorophores and their mixture enables to calculate autocorrelation functions of each of the fluorophores and thus their diffusion properties and concentrations can be determined separately. Moreover, crosscorrelation of the two signals can be performed and information on interaction of the species can be obtained. This technique is particularly helpful for distinguishing different states of the same fluorophore in different microenvironments. The first application of that concept represents the simultaneous determination of two-dimensional diffusion in planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers. The lifetime in both investigated systems differed because the lifetime of the dye is considerably quenched in the layer near the light-absorbing surface. This concept was also used in other applications: a) investigation of a conformational change of a labeled protein, b) detection of small amounts of labeled oligonucleotides bound to metal particles or c) elucidation of the compaction mechanism of different sized labeled DNA molecules. Moreover, it was demonstrated that FLCS can help to overcome some FCS experimental drawbacks.

  17. Fluorescence spectroscopy using indocyanine green for lymph node mapping

    NASA Astrophysics Data System (ADS)

    Haj-Hosseini, Neda; Behm, Pascal; Shabo, Ivan; Wârdell, Karin

    2014-02-01

    The principles of cancer treatment has for years been radical resection of the primary tumor. In the oncologic surgeries where the affected cancer site is close to the lymphatic system, it is as important to detect the draining lymph nodes for metastasis (lymph node mapping). As a replacement for conventional radioactive labeling, indocyanine green (ICG) has shown successful results in lymph node mapping; however, most of the ICG fluorescence detection techniques developed are based on camera imaging. In this work, fluorescence spectroscopy using a fiber-optical probe was evaluated on a tissue-like ICG phantom with ICG concentrations of 6-64 μM and on breast tissue from five patients. Fiber-optical based spectroscopy was able to detect ICG fluorescence at low intensities; therefore, it is expected to increase the detection threshold of the conventional imaging systems when used intraoperatively. The probe allows spectral characterization of the fluorescence and navigation in the tissue as opposed to camera imaging which is limited to the view on the surface of the tissue.

  18. Polarized fluorescence correlation spectroscopy of DNA-DAPI complexes.

    PubMed

    Barcellona, Maria Luisa; Gammon, Seth; Hazlett, Theodore; Digman, Michelle A; Gratton, Enrico

    2004-11-01

    We discuss the use of fluorescence correlation spectroscopy for the measurement of relatively slow rotations of large macromolecules in solution or attached to other macromolecular structures. We present simulations and experimental results to illustrate the range of rotational correlation times and diffusion times that the technique can analyze. In particular, we examine various methods to analyze the polarization fluctuation data. We have found that by first constructing the polarization function and then calculating the autocorrelation function, we can obtain the rotational motion of the molecule with very little interference from the lateral diffusion of the macromolecule, as long as the rotational diffusion is significantly faster than the lateral diffusion. Surprisingly, for common fluorophores the autocorrelation of the polarization function is relatively unaffected by the photon statistics. In our instrument, two-photon excitation is used to define a small volume of illumination where a few molecules are present at any instant of time. The measurements of long DNA molecules labeled with the fluorescent probe DAPI show local rotational motions of the polymers in addition to translation motions of the entire polymer. For smaller molecules such as EGFP, the viscosity of the solution must be increased to bring the relaxation due to rotational motion into the measurable range. Overall, our results show that polarized fluorescence correlation spectroscopy can be used to detect fast and slow rotational motion in the time scale from microsecond to second, a range that cannot be easily reached by conventional fluorescence anisotropy decay methods.

  19. The spectroscopic basis of Fluorescence Triple Correlation Spectroscopy

    PubMed Central

    Ridgeway, William K.; Millar, David P.; Williamson, James R.

    2012-01-01

    We have developed Fluorescence Triple Correlation Spectroscopy (F3CS) as an extension of the widely-used fluorescence microscopy technique Fluorescence Correlation Spectroscopy. F3CS correlates three signals at once and provides additional capabilities for the study of systems with complex stoichiometry, kinetic processes and irreversible reactions. A general theory of F3CS was developed to describe the interplay of molecular dynamics and microscope optics, leading to an analytical function to predict experimental triple correlations of molecules that freely diffuse through the tight focus of the microscope. Experimental correlations were calculated from raw fluorescence data using triple correlation integrals that extend multiple-tau correlation theory to delay times in two dimensions. The quality of experimental data was improved by tuning specific spectroscopic parameters and employing multiple independent detectors to minimize optoelectronic artifacts. Experiments with the reversible system of freely-diffusing 16S rRNA revealed that triple correlation functions contain symmetries predicted from time-reversal arguments. Irreversible systems are shown to break these symmetries and correlation strategies were developed to detect time-reversal asymmetries in a comprehensive way with respect to two delay times, each spanning many orders of magnitude in time. The correlation strategies, experimental approaches and theory developed here enable studies of the composition and dynamics of complex systems using F3CS. PMID:22229664

  20. Rapid discrimination of extracts of Chinese propolis and poplar buds by FT-IR and 2D IR correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Wu, Yan-Wen; Sun, Su-Qin; Zhao, Jing; Li, Yi; Zhou, Qun

    2008-07-01

    The extract of Chinese propolis (ECP) has recently been adulterated with that of poplar buds (EPB), because most of ECP is derived from the poplar plant, and ECP and EPB have almost identical chemical compositions. It is very difficult to differentiate them by using the chromatographic methods such as high performance liquid chromatography (HPLC) and gas chromatography (GC). Therefore, how to effectively discriminate these two mixtures is a problem to be solved urgently. In this paper, a rapid method for discriminating ECP and EPB was established by the Fourier transform infrared (FT-IR) spectra combined with the two-dimensional infrared correlation (2D IR) analysis. Forty-three ECP and five EPB samples collected from different areas of China were analyzed by the FT-IR spectroscopy. All the ECP and EPB samples tested show similar IR spectral profiles. The significant differences between ECP and EPB appear in the region of 3000-2800 cm -1 of the spectra. Based on such differences, the two species were successfully classified with the soft independent modeling of class analogy (SIMCA) pattern recognition technique. Furthermore, these differences were well validated by a series of temperature-dependent dynamic FT-IR spectra and the corresponding 2D IR plots. The results indicate that the differences in these two natural products are caused by the amounts of long-chain alkyl compounds (including long-chain alkanes, long-chain alkyl esters and long chain alkyl alcohols) in them, rather than the flavonoid compounds, generally recognized as the bioactive substances of propolis. There are much more long-chain alkyl compounds in ECP than those in EPB, and the carbon atoms of the compounds in ECP remain in an order Z-shaped array, but those in EPB are disorder. It suggests that FT-IR and 2D IR spectroscopy can provide a valuable method for the rapid differentiation of similar natural products, ECP and EPB. The IR spectra could directly reflect the integrated chemical

  1. Stark Spectroscopy of Rubrene. II. Stark Fluorescence Spectroscopy and Fluorescence Quenching Induced by an External Electric Field.

    PubMed

    Iimori, Toshifumi; Ito, Ryuichi; Ohta, Nobuhiro

    2016-07-21

    We report Stark fluorescence spectroscopy investigation of rubrene dispersed in a poly(methyl methacrylate) film. The features of the fluorescence spectrum are analogous to those in solutions. In the Stark fluorescence spectrum, the decrease of the fluorescence quantum yield in the presence of an external electric field is observed. This result shows that the yield of nonradiative decay processes is increased by the application of an external electric field. It is known that the fluorescence quantum yield for rubrene, which is nearly unity at room temperature, depends on temperature, and a major nonradiative decay process in photoexcited rubrene is ascribed to a thermally activated intersystem crossing (ISC). Equations that express the field-induced fluorescence quenching in terms of the molecular parameters are derived from the ensemble average of electric field effects on the activation energy of the reaction rate constant in random orientation systems. The molecular parameters are then extracted from the observed data. It is inferred that the field-induced increase in the yield of other intramolecular and intermolecular photophysical processes in addition to the ISC should be taken into account.

  2. Accounting for misalignments and thermal fluctuations in fluorescence correlation spectroscopy experiments on membranes.

    PubMed

    Sanguigno, Luigi; Cosenza, Chiara; Causa, Filippo; Netti, Paolo Antonio

    2013-03-21

    Several authors have exploited the ability of the fluorescence correlation spectroscopy to probe motion at the molecular level. In a couple of decades, all their efforts have allowed the application of this technique even to the diffusion measurement of cellular components. Nowadays, the fluorescence correlation spectroscopy is considered a standard tool to measure diffusion in cells both in vivo and in vitro. Unfortunately, while the interpretation and the set-up have been consolidated for 3D diffusion measurements (i.e. diffusion in an aqueous solution), the experiments carried out on flat elements, such as membranes, show unusually high relative errors. Furthermore, long tail correlations are generally detected and ascribed to diffusion anomalies. The 2D fluorescence correlation measurements have been interpreted under certain hypotheses, whereby the membrane is assumed to be perfectly flat, motionless and aligned with the optical axes. Here, we investigated the robustness of these hypotheses, trying to understand, in an elementary but not trivial way, how misalignments and thermal fluctuations affect the temporal correlation of the intensity fluctuation collected during measurements on membranes.

  3. Synchronous fluorescence spectroscopy for analysis of wine and wine distillates

    NASA Astrophysics Data System (ADS)

    Andreeva, Ya.; Borisova, E.; Genova, Ts.; Zhelyazkova, Al.; Avramov, L.

    2015-01-01

    Wine and brandies are multicomponent systems and conventional fluorescence techniques, relying on recording of single emission or excitation spectra, are often insufficient. In such cases synchronous fluorescence spectra can be used for revealing the potential of the fluorescence techniques. The technique is based on simultaneously scanning of the excitation and emission wavelength with constant difference (Δλ) maintained between them. In this study the measurements were made using FluoroLog3 spectrofluorimeter (HORIBA Jobin Yvon, France) and collected for excitation and emission in the wavelength region 220 - 700 nm using wavelength interval Δλ from 10 to 100 nm in 10 nm steps. This research includes the results obtained for brandy and red wine samples. Fluorescence analysis takes advantage in the presence of natural fluorophores in wines and brandies, such as gallic, vanillic, p-coumaric, syringic, ferulic acid, umbelliferone, scopoletin and etc. Applying of synchronous fluorescence spectroscopy for analysis of these types of alcohols allows us to estimate the quality of wines and also to detect adulteration of brandies like adding of a caramel to wine distillates for imitating the quality of the original product aged in oak casks.

  4. Lifetime fluorescence spectroscopy for in situ investigation of osteogenic differentiation

    NASA Astrophysics Data System (ADS)

    Marcu, Laura; Elbarbary, Amir; Zuk, Patricia; De Ugarte, Daniel A.; Benhaim, Prosper; Kurt, Hamza; Hedrick, Marc H.; Ashjian, Peter

    2003-07-01

    Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) represents a potential tool for the in-situ characterization of bioengineered tissues. In this study, we evaluate the application of TR-LIFS to non-intrusive monitoring of matrix composition during osteogenetic differentiation. Human adipose-derived stem cells, harvested from 3 patients, were induced in osteogenic media for 3, 5, and 7 weeks. Samples were subsequently collected and probed for time-resolved fluorescence emission with a pulsed nitrogen laser. Fluorescence parameters, derived from both spectral- and time-domain, were used for sample characterization. The samples were further analyzed using Western blot analysis and computer-based densitometry. A significant change in the fluorescence parameters was detected for samples beyond 3 weeks of osteogenic differentiation. The spectroscopic observations: 1) show increase of collagen I when contrasted against the time-resolved fluorescence spectra of commercially available collagens; and 2) are in agreement with Western blot analysis that demonstrated significant increase in collagen I content between 3- vs. 5-weeks and 3- vs. 7-weeks and no changes for collagens III, IV, and V. Our results suggest that TR-LIFS can be used as a non-invasive means for the detection of specific collagens in maturing connective tissues.

  5. In ovo sexing of chicken eggs by fluorescence spectroscopy.

    PubMed

    Galli, Roberta; Preusse, Grit; Uckermann, Ortrud; Bartels, Thomas; Krautwald-Junghanns, Maria-Elisabeth; Koch, Edmund; Steiner, Gerald

    2017-02-01

    Culling of day-old male chicks in production of laying hen strains involves several millions of animals every year worldwide and is ethically controversial. In an attempt to provide an alternative, optical spectroscopy was investigated to determine nondestructively in ovo the sex of early embryos of the domestic chicken. The extraembryonic blood circulation system was accessed by producing a window in the egg shell and the flowing blood was illuminated with a near-infrared laser. The strong fluorescence and the weak Raman signals were acquired and spectroscopically analyzed between 800 and 1000 nm. The increase of fluorescence intensity between 3.5 and 11.5 days of incubation was found to be in agreement with the erythropoietic stages, thus enabling to identify hemoglobin as fluorescence source. Sex-related differences in the fluorescence spectrum were found at day 3.5, and principal component (PC) analysis showed that the blood of males was characterized by a specific fluorescence band located at ∼910 nm. Supervised classification of the PC scores enabled the determination of the sex of 380 eggs at day 3.5 of incubation with a correct rate up to 93% by combining the information derived from both fluorescence and Raman scattering. Graphical abstract The fluorescence of blood obtained in ovo by illumination of embryonic vessels with a IR laser displays spectral differences that can be employed for sexing of eggs in early stage of incubation, before onset of embryo sensitivity and without hindering its development into a healthy chick.

  6. Fluorescence Spectroscopy of Gas-phase Polycyclic Aromatic Hydrocarbons

    NASA Technical Reports Server (NTRS)

    Thomas, J. D.; Witt, A. N.

    2006-01-01

    The purpose of this investigation was to produce fluorescence spectra of polycyclic aromatic hydrocarbon (PAH) molecules in the gas-phase for comparison with blue luminescence (BL) emission observed in astrophysical sources Vijh et al. (2004, 2005a,b). The BL occurs roughly from 350 to 450 nm, with a sharp peak near 380 nm. PAHs with three to four rings, e.g. anthracene and pyrene, were found to produce luminescence in the appropriate spectral region, based on existing studies. Relatively few studies of the gas-phase fluorescence of PAHs exist; those that do exist have dealt primarily with the same samples commonly available for purchase such as pyrene and anthracene. In an attempt to understand the chemistry of the nebular environment we also obtained several nitrogen substituted PAHs from our colleagues at NASA Ames. In order to simulate the astrophysical environment we also took spectra by heating the PAHs in a flame. The flame environment counteracts the formation of eximers and permits the spectroscopy of free-flying neutral molecules. Experiments with coal tar demonstrate that fluorescence spectroscopy reveals primarily the presence of the smallest molecules, which are most abundant and which possess the highest fluorescence efficiencies. One gas-phase PAH that seems to fit the BL spectrum most closely is phenanthridine. In view of the results from the spectroscopy of coal tar, a compound containing a mixture of PAHs ranging from small to very large PAH molecules, we can not preclude the presence of larger PAHs in interstellar sources exhibiting BL.

  7. Fluorescent rare earth solutions as intrinsic wavelength standards for protein fluorescence spectroscopy.

    PubMed

    Anderle, Heinz; Weber, Alfred

    2017-02-01

    Trivalent Gd, Tm, and Dy solutions can be used as intrinsic excitation and emission standards to validate the UV and violet-blue wavelength accuracy of a spectrofluorimeter. Europium extends the range into the red. To attain sufficient sensitivity, these luminescent rare earth ions require deuterated reagents or carbonate complexation, which allow the use of ordinary water and thus preparation in virtually any laboratory. Such solutions are particularly valuable as system suitability standards (SST) for protein fluorescence spectroscopy to detect red shifts of the intrinsic fluorescence maximum in stability and storage studies.

  8. Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging

    PubMed Central

    Yankelevich, Diego R.; Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Elson, Daniel S.; Marcu, Laura

    2014-01-01

    The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8–7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence

  9. Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging

    SciTech Connect

    Yankelevich, Diego R.; Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Marcu, Laura; Elson, Daniel S.

    2014-03-15

    The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8–7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence

  10. Optimized Time-Gated Fluorescence Spectroscopy for the Classification and Recycling of Fluorescently Labeled Plastics.

    PubMed

    Fomin, Petr; Zhelondz, Dmitry; Kargel, Christian

    2016-08-29

    For the production of high-quality parts from recycled plastics, a very high purity of the plastic waste to be recycled is mandatory. The incorporation of fluorescent tracers ("markers") into plastics during the manufacturing process helps overcome typical problems of non-tracer based optical classification methods. Despite the unique emission spectra of fluorescent markers, the classification becomes difficult when the host plastics exhibit (strong) autofluorescence that spectrally overlaps the marker fluorescence. Increasing the marker concentration is not an option from an economic perspective and might also adversely affect the properties of the plastics. A measurement approach that suppresses the autofluorescence in the acquired signal is time-gated fluorescence spectroscopy (TGFS). Unfortunately, TGFS is associated with a lower signal-to-noise (S/N) ratio, which results in larger classification errors. In order to optimize the S/N ratio we investigate and validate the best TGFS parameters-derived from a model for the fluorescence signal-for plastics labeled with four specifically designed fluorescent markers. In this study we also demonstrate the implementation of TGFS on a measurement and classification prototype system and determine its performance. Mean values for a sensitivity of [Formula: see text] = 99.93% and precision [Formula: see text] = 99.80% were achieved, proving that a highly reliable classification of plastics can be achieved in practice.

  11. Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

    2015-07-01

    Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

  12. Cross Talk Free Fluorescence Cross Correlation Spectroscopy in Live Cells

    PubMed Central

    Thews, Elmar; Gerken, Margarita; Eckert, Reiner; Zäpfel, Johannes; Tietz, Carsten; Wrachtrup, Jörg

    2005-01-01

    Fluorescence correlation spectroscopy (FCS) is now a widely used technique to measure small ensembles of labeled biomolecules with single molecule detection sensitivity (e.g., low endogenous concentrations). Fluorescence cross correlation spectroscopy (FCCS) is a derivative of this technique that detects the synchronous movement of two biomolecules with different fluorescence labels. Both methods can be applied to live cells and, therefore, can be used to address a variety of unsolved questions in cell biology. Applications of FCCS with autofluorescent proteins (AFPs) have been hampered so far by cross talk between the detector channels due to the large spectral overlap of the fluorophores. Here we present a new method that combines advantages of these techniques to analyze binding behavior of proteins in live cells. To achieve this, we have used dual color excitation of a common pair of AFPs, ECFP and EYFP, being discriminated in excitation rather than in emission. This is made possible by pulsed excitation and detection on a shorter timescale compared to the average residence time of particles in the FCS volume element. By this technique we were able to eliminate cross talk in the detector channels and obtain an undisturbed cross correlation signal. The setup was tested with ECFP/EYFP lysates as well as chimeras as negative and positive controls and demonstrated to work in live HeLa cells coexpressing the two fusion proteins ECFP-connexin and EYFP-connexin. PMID:15951373

  13. Two-dimensional fluorescence spectroscopy of laser-produced plasmas

    SciTech Connect

    Harilal, Sivanandan S.; LaHaye, Nicole L.; Phillips, Mark C.

    2016-08-01

    We use a two-dimensional laser-induced fluorescence spectroscopy technique to measure the coupled absorption and emission properties of atomic species in plasmas produced via laser ablation of solid aluminum targets at atmospheric pressure. Emission spectra from the Al I 394.4 nm and Al I 396.15 nm transitions are measured while a frequency-doubled, continuous-wave, Ti:Sapphire laser is tuned across the Al I 396.15 nm transition. The resulting two-dimensional spectra show the energy coupling between the two transitions via increased emission intensity for both transitions during resonant absorption of the continuous-wave laser at one transition. Time-delayed and gated detection of the emission spectrum is used to isolate the resonantly-excited fluorescence emission from the thermally-excited emission from the plasma. In addition, the tunable continuous-wave laser measures the absorption spectrum of the Al transition with ultra-high resolution after the plasma has cooled, resulting in narrower spectral linewidths than observed in emission spectra. Our results highlight that fluorescence spectroscopy employing continuous-wave laser re-excitation after pulsed laser ablation combines benefits of both traditional emission and absorption spectroscopic methods.

  14. Energy transfer dynamics in trimers and aggregates of light-harvesting complex II probed by 2D electronic spectroscopy

    SciTech Connect

    Enriquez, Miriam M.; Zhang, Cheng; Tan, Howe-Siang; Akhtar, Parveen; Garab, Győző; Lambrev, Petar H.

    2015-06-07

    The pathways and dynamics of excitation energy transfer between the chlorophyll (Chl) domains in solubilized trimeric and aggregated light-harvesting complex II (LHCII) are examined using two-dimensional electronic spectroscopy (2DES). The LHCII trimers and aggregates exhibit the unquenched and quenched excitonic states of Chl a, respectively. 2DES allows direct correlation of excitation and emission energies of coupled states over population time delays, hence enabling mapping of the energy flow between Chls. By the excitation of the entire Chl b Q{sub y} band, energy transfer from Chl b to Chl a states is monitored in the LHCII trimers and aggregates. Global analysis of the two-dimensional (2D) spectra reveals that energy transfer from Chl b to Chl a occurs on fast and slow time scales of 240–270 fs and 2.8 ps for both forms of LHCII. 2D decay-associated spectra resulting from the global analysis identify the correlation between Chl states involved in the energy transfer and decay at a given lifetime. The contribution of singlet–singlet annihilation on the kinetics of Chl energy transfer and decay is also modelled and discussed. The results show a marked change in the energy transfer kinetics in the time range of a few picoseconds. Owing to slow energy equilibration processes, long-lived intermediate Chl a states are present in solubilized trimers, while in aggregates, the population decay of these excited states is significantly accelerated, suggesting that, overall, the energy transfer within the LHCII complexes is faster in the aggregated state.

  15. Energy transfer dynamics in trimers and aggregates of light-harvesting complex II probed by 2D electronic spectroscopy.

    PubMed

    Enriquez, Miriam M; Akhtar, Parveen; Zhang, Cheng; Garab, Győző; Lambrev, Petar H; Tan, Howe-Siang

    2015-06-07

    The pathways and dynamics of excitation energy transfer between the chlorophyll (Chl) domains in solubilized trimeric and aggregated light-harvesting complex II (LHCII) are examined using two-dimensional electronic spectroscopy (2DES). The LHCII trimers and aggregates exhibit the unquenched and quenched excitonic states of Chl a, respectively. 2DES allows direct correlation of excitation and emission energies of coupled states over population time delays, hence enabling mapping of the energy flow between Chls. By the excitation of the entire Chl b Qy band, energy transfer from Chl b to Chl a states is monitored in the LHCII trimers and aggregates. Global analysis of the two-dimensional (2D) spectra reveals that energy transfer from Chl b to Chl a occurs on fast and slow time scales of 240-270 fs and 2.8 ps for both forms of LHCII. 2D decay-associated spectra resulting from the global analysis identify the correlation between Chl states involved in the energy transfer and decay at a given lifetime. The contribution of singlet-singlet annihilation on the kinetics of Chl energy transfer and decay is also modelled and discussed. The results show a marked change in the energy transfer kinetics in the time range of a few picoseconds. Owing to slow energy equilibration processes, long-lived intermediate Chl a states are present in solubilized trimers, while in aggregates, the population decay of these excited states is significantly accelerated, suggesting that, overall, the energy transfer within the LHCII complexes is faster in the aggregated state.

  16. Investigation on the effect of fluorescence quenching of bovine serum albumin by cefoxitin sodium using fluorescence spectroscopy and synchronous fluorescence spectroscopy.

    PubMed

    Li, Gaixia; Liu, Bao-Sheng; Zhang, Qiuju; Han, Rong

    2016-08-01

    The reaction mechanism of cefoxitin sodium with bovine serum albumin was investigated using fluorescence spectroscopy and synchronous fluorescence spectroscopy at different temperatures. The results showed that the change of binding constant of the synchronous fluorescence method with increasing temperature could be used to estimate the types of quenching mechanisms of drugs with protein and was consistent with one of fluorescence quenching method. In addition, the number of binding sites, type of interaction force, cooperativity between drug and protein and energy-transfer parameters of cefoxitin sodium and bovine serum albumin obtained from two methods using the same equation were consistent. Electrostatic force played a major role in the conjugation reaction between bovine serum albumin and cefoxitin sodium, and the type of quenching was static quenching. The primary binding site for cefoxitin sodium was sub-hydrophobic domain IIA, and the number of binding sites was 1. The value of Hill's coefficients (nH ) was approximately equal to 1, which suggested no cooperativity in the bovine serum albumin-cefoxitin sodium system. The donor-to-acceptor distance r < 7 nm indicated that static fluorescence quenching of bovine serum albumin by cefoxitin sodium was also a non-radiation energy-transfer process. The results indicated that synchronous fluorescence spectrometry could be used to study the reaction mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy.

    PubMed

    Widengren, Jerker; Chmyrov, Andriy; Eggeling, Christian; Löfdahl, Per-Ake; Seidel, Claus A M

    2007-01-25

    Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes

  18. Spectral decomposition of NAD(P)H fluorescence components recorded by multi-wavelength fluorescence lifetime spectroscopy in living cardiac cells

    NASA Astrophysics Data System (ADS)

    Chorvatova, Alzbeta; Mateasik, Anton; Chorvat, Dusan, Jr.

    2013-12-01

    We report a novel analytical approach to identify individual components of a cell’s endogenous fluorescence, recorded by spectrally-resolved time-correlated single photon counting (TCSPC). Time-resolved area-normalized emission spectroscopy (TRANES) and principal component analysis (PCA) were applied to estimate the number of spectral components after metabolic modulation of cardiac cells following excitation with a 375 nm picosecond laser. Linear unmixing of TCSPC data spectrally decomposed individual components in living cells, while using characteristics of endogenously fluorescing molecules in solvents as a reference spectral database. Our data demonstrate the presence of three individual components, corresponding to the nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in organic and inorganic solvents and to the residual flavoprotein fluorescence. The presented analytical approach offers a new alternative for the spectral separation of multi-wavelength fluorescence lifetime spectroscopy data to the conventional analysis, and opens a new possibility for the use of pattern recognition for fast resolution of components in 2D fluorescence lifetime microscopy images.

  19. An analog filter approach to frequency domain fluorescence spectroscopy

    DOE PAGES

    Trainham, Clifford P.; O'Neill, Mary D.; McKenna, Ian J.

    2015-10-01

    The rate equations found in frequency domain fluorescence spectroscopy are the same as those found in electronics under analog filter theory. Laplace transform methods are a natural way to solve the equations, and the methods can provide solutions for arbitrary excitation functions. The fluorescence terms can be modeled as circuit components and cascaded with drive and detection electronics to produce a global transfer function. Electronics design tools such as Spicea can be used to model fluorescence problems. In applications, such as remote sensing, where detection electronics are operated at high gain and limited bandwidth, a global modeling of the entiremore » system is important, since the filter terms of the drive and detection electronics affect the measured response of the fluorescence signals. Furthermore, the techniques described here can be used to separate signals from fast and slow fluorophores emitting into the same spectral band, and data collection can be greatly accelerated by means of a frequency comb driver waveform and appropriate signal processing of the response.« less

  20. An analog filter approach to frequency domain fluorescence spectroscopy

    SciTech Connect

    Trainham, Clifford P.; O'Neill, Mary D.; McKenna, Ian J.

    2015-10-01

    The rate equations found in frequency domain fluorescence spectroscopy are the same as those found in electronics under analog filter theory. Laplace transform methods are a natural way to solve the equations, and the methods can provide solutions for arbitrary excitation functions. The fluorescence terms can be modeled as circuit components and cascaded with drive and detection electronics to produce a global transfer function. Electronics design tools such as Spicea can be used to model fluorescence problems. In applications, such as remote sensing, where detection electronics are operated at high gain and limited bandwidth, a global modeling of the entire system is important, since the filter terms of the drive and detection electronics affect the measured response of the fluorescence signals. Furthermore, the techniques described here can be used to separate signals from fast and slow fluorophores emitting into the same spectral band, and data collection can be greatly accelerated by means of a frequency comb driver waveform and appropriate signal processing of the response.

  1. Brain cancer probed by native fluorescence and stokes shift spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhou, Yan; Liu, Cheng-hui; He, Yong; Pu, Yang; Li, Qingbo; Wang, Wei; Alfano, Robert R.

    2012-12-01

    Optical biopsy spectroscopy was applied to diagnosis human brain cancer in vitro. The spectra of native fluorescence, Stokes shift and excitation spectra were obtained from malignant meningioma, benign, normal meningeal tissues and acoustic neuroma benign tissues. The wide excitation wavelength ranges were used to establish the criterion for distinguishing brain diseases. The alteration of fluorescence spectra between normal and abnormal brain tissues were identified by the characteristic fluorophores under the excitation with UV to visible wavelength range. It was found that the ratios of the peak intensities and peak position in both spectra of fluorescence and Stokes shift may be used to diagnose human brain meninges diseases. The preliminary analysis of fluorescence spectral data from cancer and normal meningeal tissues by basic biochemical component analysis model (BBCA) and Bayes classification model based on statistical methods revealed the changes of components, and classified the difference between cancer and normal human brain meningeal tissues in a predictions accuracy rate is 0.93 in comparison with histopathology and immunohistochemistry reports (gold standard).

  2. Optimal algorithm for fluorescence suppression of modulated Raman spectroscopy.

    PubMed

    Mazilu, Michael; De Luca, Anna Chiara; Riches, Andrew; Herrington, C Simon; Dholakia, Kishan

    2010-05-24

    Raman spectroscopy permits probing of the molecular and chemical properties of the analyzed sample. However, its applicability has been seriously limited to specific applications by the presence of a strong fluorescence background. In our recent paper [Anal. Chem. 82, 738 (2010)], we reported a new modulation method for separating Raman scattering from fluorescence. By continuously changing the excitation wavelength, we demonstrated that it is possible to continuously shift the Raman peaks while the fluorescence background remains essentially constant. In this way, our method allows separation of the modulated Raman peaks from the static fluorescence background with important advantages when compared to previous work using only two [Appl. Spectrosc. 46, 707 (1992)] or a few shifted excitation wavelengths [Opt. Express 16, 10975 (2008)]. The purpose of the present work is to demonstrate a significant improvement of the efficacy of the modulated method by using different processing algorithms. The merits of each algorithm (Standard Deviation analysis, Fourier Filtering, Least-Squares fitting and Principal Component Analysis) are discussed and the dependence of the modulated Raman signal on several parameters, such as the amplitude and the modulation rate of the Raman excitation wavelength, is analyzed. The results of both simulation and experimental data demonstrate that Principal Component Analysis is the best processing algorithm. It improves the signal-to-noise ratio in the treated Raman spectra, reducing required acquisition times. Additionally, this approach does not require any synchronization procedure, reduces user intervention and renders it suitable for real-time applications.

  3. Investigation of asphaltene association by front-face fluorescence spectroscopy.

    PubMed

    Albuquerque, Flávio Cortiñas; Nicodem, David E; Rajagopal, Krishnaswamy

    2003-07-01

    The tendency of asphaltenes to aggregate and form clusters in solvents was studied by fluorescence spectroscopy. This was done by evaluating the relative fluorescence quantum yield of asphaltenes diluted at several concentrations in toluene and by studying the changes in the fluorescence spectra of asphaltene solutions as the composition of the solvent, toluene and cyclohexane, is changed. The asphaltene fraction (heptane insoluble) was collected from a Brazilian heavy crude oil, and solutions of this material varying from 0.016 g/L up to 10 g/L were prepared in toluene. Front-face emission spectra were obtained in two wavelength ranges, from 310 to 710 nm, excited at 300 nm (short range), and from 410 to 710 nm, excited at 400 nm (long range). Severe quenching was observed at concentrations above about 0.1 g/L. Stern-Volmer plots (reciprocal of quantum yield against concentration) exhibited nonlinear, downward-curved behavior, indicating that a more complex suppression mechanism, probably influenced by the association of the asphaltene molecules, is taking place. The same asphaltenes were dissolved (0.1 g/L) in binary mixtures of toluene and cyclohexane, and emission spectra in both the short range and long range were obtained. Fluorescence was progressively quenched at longer wavelengths of the spectra as the proportion of cyclohexane in the solvent grew. Cyclohexane, a poor asphaltene solvent, is probably inducing static quenching through association of asphaltenes.

  4. Fluorescence spectroscopy for endogenous porphyrins in human facial skin

    NASA Astrophysics Data System (ADS)

    Seo, I.; Tseng, S. H.; Cula, G. O.; Bargo, P. R.; Kollias, N.

    2009-02-01

    The activity of certain bacteria in skin is known to correlate to the presence of porphyrins. In particular the presence of coproporphyrin produced by P.acnes inside plugged pores has been correlated to acne vulgaris. Another porphyrin encountered in skin is protoporphyrin IX, which is produced by the body in the pathway for production of heme. In the present work, a fluorescence spectroscopy system was developed to measure the characteristic spectrum and quantify the two types of porphyrins commonly present in human facial skin. The system is comprised of a Xe lamp both for fluorescence excitation and broadband light source for diffuse reflectance measurements. A computer-controlled filter wheel enables acquisition of sequential spectra, first excited by blue light at 405 nm then followed by the broadband light source, at the same location. The diffuse reflectance spectrum was used to correct the fluorescence spectrum due to the presence of skin chromophores, such as blood and melanin. The resulting fluorescence spectra were employed for the quantification of porphyrin concentration in a population of healthy subjects. The results show great variability on the concentration of these porphyrins and further studies are being conducted to correlate them with skin conditions such as inflammation and acne vulgaris.

  5. Biosensors based on surface plasmon-enhanced fluorescence spectroscopy.

    PubMed

    Dostálek, Jakub; Knoll, Wolfgang

    2008-09-01

    The implementation of surface plasmon-enhanced fluorescence spectroscopy (SPFS) to surface plasmon resonance (SPR) biosensors enables increasing their sensitivity by several orders of magnitude. In SPR-based biosensors, surface plasmons probe the binding of target molecules contained in a liquid sample by their affinity partners attached to a metallic sensor surface. SPR biosensors relying on the detection of refractive index changes allow for direct observation of the binding of large and medium size molecules that produces sufficiently large refractive index changes. In SPR biosensors exploiting SPFS, the capture of fluorophore-labeled molecules to the sensor surface is observed by the detection of fluorescence light emitted from the surface. This technique takes advantage of the enhanced intensity of electromagnetic field accompanied with the resonant excitation of surface plasmons. The interaction with surface plasmons can greatly increase the measured fluorescence signal through enhancing the excitation rate of fluorophores and by more efficient collecting of fluorescence light. SPFS-based biosensors were shown to enable the analysis of samples with extremely low analyte concentrations and the detection of small molecules. In this review, we describe the fundamental principles, implementations, and current state of the art applications of SPFS biosensors. This review focuses on SPFS-based biosensors employing the excitation of surface plasmons on continuous metal-dielectric interfaces.

  6. The study of blue LED to induce fluorescence spectroscopy and fluorescence imaging for oral carcinoma detection

    NASA Astrophysics Data System (ADS)

    Zheng, Longjiang; Hu, Yuanting

    2009-07-01

    Fluorescence spectroscopy and fluorescence imaging diagnosis of malignant lesions provides us with a new method to diagnose diseases in precancerous stage. Early diagnosis of disease has significant importance in cancer treatment, because most cancers can be cured well in precancerous, especially when the diffusion of cancer is limited in a restricted region. In this study, Golden hamster models were applied to 5% 9, 10 dimethyl-1, 2-benzanthracene (DMBA) to induce hamster buccal cheek pouch carcinoma three times a week. Rose Bengal, which has been used in clinican for years and avoids visible side-effect to human was chosen as photosensitizer. 405 nm blue LED was used to induce the fluorescence of photosensitizer. After topical application of photosensitizer, characteristic red emission fluorescence peak was observed around 600nm. Similar, normal oral cavity has special luminescence around 480nm. Fluorescence spectroscopy technology is based on analysing emission peaks of photosensitizer in the areas of oral carcinoma, moreover, red-to-green (IR/IG) intensity ratio is also applied as a diagnostic algorithm. A CCD which is connected with a computer is used to take pictures at carcinoma areas through different filters. Fluorescence images from normal hamster buccal cheek pouch are compared with those from carcinogen-induced models of carcinoma, and morphological differences between normal and lesion tissue can be distinguished. The pictures are analyzed by Matlab and shown on the screen of computer. This paper demonstrates that Rose Bengal could be used as photosensitizer to detect oral carcinoma, and blue LED as excitation source could not only have a good effect to diagnose oral carcinoma, but also decrease cost greatly.

  7. Two-Photon-Excited Fluorescence-Encoded Infrared Spectroscopy.

    PubMed

    Mastron, Joseph N; Tokmakoff, Andrei

    2016-11-23

    We report on a method for performing ultrafast infrared (IR) vibrational spectroscopy using fluorescence detection. Vibrational dynamics on the ground electronic state driven by femtosecond mid-infrared pulses are detected by changes in fluorescence amplitude resulting from modulation of a two-photon visible transition by nuclear motion. We examine a series of coumarin dyes and study the signals as a function of solvent and excitation pulse parameters. The measured signal characterizes the relaxation of vibrational populations and coherences but yields different information than conventional IR transient absorption measurements. These differences result from the manner in which the ground-state dynamics are projected by the two-photon detection step. Extensions of this method can be adapted for a variety of increased-sensitivity IR measurements.

  8. Influence of the surface hydrophobicity on fluorescence correlation spectroscopy measurements

    NASA Astrophysics Data System (ADS)

    Boutin, Céline; Jaffiol, Rodolphe; Plain, Jérome; Royer, Pascal

    2007-02-01

    Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique used to analyze the diffusion at the single molecule level in solution. FCS is based on the temporal autocorrelation of fluorescent signal generated by dye molecules diffusing through a small confocal volume. These measurements are mostly carried out in a chambered coverglass, close to the glass substrate. In this report, we discuss how the chemical nature of the glass-water interface may interact with the free diffusion of molecules. Our results reveal a strong influence, up to a few μm from the interface, of the surface hydrophobicity degree. This influence is assessed through the relative weight of the two dimension diffusion process observed at the vicinity of the surface.

  9. [Identification and analysis of genuine and false Flos Rosae Rugosae by FTIR and 2D correlation IR spectroscopy].

    PubMed

    Cai, Fang; Sun, Su-qin; Yan, Wen-rong; Niu, Shi-jie; Li, Xian-en

    2009-09-01

    The genuine and false Flos Rosae Rugosae (Flos Rosae Chinensis and Flos Rosa multiflora) were examined in terms of their differences by using Fourier transform infrared spectroscopy (FTIR) combined with two-dimensional (2D) correlation IR spectroscopy. The three species were shown very similar in FTIR spectra. The peak of 1318 cm(-1) of genuine Flos Rosae Rugosae is not obvious but this peak could be found sharp in Flos Rosae Chinensis and Flos Rosa multiflora. Generally, the second derivative IR spectrum can clearly enhance the spectral resolution. Flos Rosae Rugosae and Flos rosae Chinensis have aromatic compounds distinct fingerprint characteristics at 1 617 and 1 618 cm(-1), respectively. Nevertheless, FlosRosa multiflora has the peak at 1612 cm(-1). There is a discrepancy of 5 to 6 cm(-1). FlosRosa multiflora has glucide's distinct fingerprint characteristics at 1 044 cm(-1), but Flos Rosae Rugosae and Flos Rosae Chinensis don't. The second derivative infrared spectra indicated different fingerprint characteristics. Three of them showed aromatic compounds with autopeaks at 1620, 1560 and 1460 cm(-1). Flos Rosae Chinensis and Flos Rosa multiflora have the shoulder peak at 1660 cm(-1). In the range of 850-1250 cm(-1), three of them are distinct different, Flos Rosae Rugosae has the strongest autopeak, Flos Rosae Chinensis has the feeble autopeak and Flos Rosa multiflora has no autopeak at 1050 cm(-1). In third-step identification, the different contents of aromatic compounds and glucide in Flos Rosae Rugosae, Flos Rosae Chinensis and Flos Rosa multiflora were revealed. It is proved that the method is fast and effective for distinguishing and analyzing genuine Flos Rosae Rugosae and false Flos Rosae Rugosae (Flos Rosae Chinensis and Flos Rosa multiflora).

  10. Highly-accelerated quantitative 2D and 3D localized spectroscopy with linear algebraic modeling (SLAM) and sensitivity encoding

    NASA Astrophysics Data System (ADS)

    Zhang, Yi; Gabr, Refaat E.; Zhou, Jinyuan; Weiss, Robert G.; Bottomley, Paul A.

    2013-12-01

    Noninvasive magnetic resonance spectroscopy (MRS) with chemical shift imaging (CSI) provides valuable metabolic information for research and clinical studies, but is often limited by long scan times. Recently, spectroscopy with linear algebraic modeling (SLAM) was shown to provide compartment-averaged spectra resolved in one spatial dimension with many-fold reductions in scan-time. This was achieved using a small subset of the CSI phase-encoding steps from central image k-space that maximized the signal-to-noise ratio. Here, SLAM is extended to two- and three-dimensions (2D, 3D). In addition, SLAM is combined with sensitivity-encoded (SENSE) parallel imaging techniques, enabling the replacement of even more CSI phase-encoding steps to further accelerate scan-speed. A modified SLAM reconstruction algorithm is introduced that significantly reduces the effects of signal nonuniformity within compartments. Finally, main-field inhomogeneity corrections are provided, analogous to CSI. These methods are all tested on brain proton MRS data from a total of 24 patients with brain tumors, and in a human cardiac phosphorus 3D SLAM study at 3T. Acceleration factors of up to 120-fold versus CSI are demonstrated, including speed-up factors of 5-fold relative to already-accelerated SENSE CSI. Brain metabolites are quantified in SLAM and SENSE SLAM spectra and found to be indistinguishable from CSI measures from the same compartments. The modified reconstruction algorithm demonstrated immunity to maladjusted segmentation and errors from signal heterogeneity in brain data. In conclusion, SLAM demonstrates the potential to supplant CSI in studies requiring compartment-average spectra or large volume coverage, by dramatically reducing scan-time while providing essentially the same quantitative results.

  11. Optical fiber fluorescence spectroscopy for detecting AFM1 in milk

    NASA Astrophysics Data System (ADS)

    Mignani, A. G.; Cucci, C.; Ciaccheri, L.; Dall'Asta, C.; Galaverna, G.; Dossena, A.; Marchelli, R.

    2008-04-01

    Fluorescence spectroscopy carried out by means of optical fibers was used for the rapid screening of M1 aflatoxin in milk, enabling the detection of concentrations up to the legal limit, which is 50 ppt. A compact fluorometric device equipped with a LED source, a miniaturized spectrometer, and optical fibers for illumination/detection of the measuring micro-cell was tested for measuring threshold values of AFM1 in pre-treated milk samples. Multivariate processing of the spectral data made it possible to obtain a preliminary screening at the earlier stages of the industrial process, as well as to discard contaminated milk stocks before their inclusion in the production chain.

  12. Identification of active fluorescence stained bacteria by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Krause, Mario; Beyer, Beatrice; Pietsch, Christian; Radt, Benno; Harz, Michaela; Rösch, Petra; Popp, Jürgen

    2008-04-01

    Microorganisms can be found everywhere e.g. in food both as useful ingredients or harmful contaminations causing food spoilage. Therefore, a fast and easy to handle analysis method is needed to detect bacteria in different kinds of samples like meat, juice or air to decide if the sample is contaminated by harmful microorganisms. Conventional identification methods in microbiology require always cultivation and therefore are time consuming. In this contribution we present an analysis approach to identify fluorescence stained bacteria on strain level by means of Raman spectroscopy. The stained bacteria are highlighted and can be localized easier against a complex sample environment e.g. in food. The use of Raman spectroscopy in combination with chemometrical methods allows the identification of single bacteria within minutes.

  13. Detectors for single-molecule fluorescence imaging and spectroscopy

    PubMed Central

    MICHALET, X.; SIEGMUND, O.H.W.; VALLERGA, J.V.; JELINSKY, P.; MILLAUD, J.E.; WEISS, S.

    2010-01-01

    Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy. PMID:20157633

  14. Fluorescence-excitation and Emission Spectroscopy on Single FMO Complexes

    PubMed Central

    Löhner, Alexander; Ashraf , Khuram; Cogdell, Richard J.; Köhler, Jürgen

    2016-01-01

    In green-sulfur bacteria sunlight is absorbed by antenna structures termed chlorosomes, and transferred to the RC via the Fenna-Matthews-Olson (FMO) complex. FMO consists of three monomers arranged in C3 symmetry where each monomer accommodates eight Bacteriochlorophyll a (BChl a) molecules. It was the first pigment-protein complex for which the structure has been determined with high resolution and since then this complex has been the subject of numerous studies both experimentally and theoretically. Here we report about fluorescence-excitation spectroscopy as well as emission spectroscopy from individual FMO complexes at low temperatures. The individual FMO complexes are subjected to very fast spectral fluctuations smearing out any possible different information from the ensemble data that were recorded under the same experimental conditions. In other words, on the time scales that are experimentally accessible by single-molecule techniques, the FMO complex exhibits ergodic behaviour. PMID:27545197

  15. The photon counting histogram in fluorescence fluctuation spectroscopy.

    PubMed Central

    Chen, Y; Müller, J D; So, P T; Gratton, E

    1999-01-01

    Fluorescence correlation spectroscopy (FCS) is generally used to obtain information about the number of fluorescent particles in a small volume and the diffusion coefficient from the autocorrelation function of the fluorescence signal. Here we demonstrate that photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence fluctuation data, i.e., the measured photon counts per molecule and the average number of molecules within the observation volume. The photon counting histogram of fluorescence fluctuation experiments, in which few molecules are present in the excitation volume, exhibits a super-Poissonian behavior. The additional broadening of the PCH compared to a Poisson distribution is due to fluorescence intensity fluctuations. For diffusing particles these intensity fluctuations are caused by an inhomogeneous excitation profile and the fluctuations in the number of particles in the observation volume. The quantitative relationship between the detected photon counts and the fluorescence intensity reaching the detector is given by Mandel's formula. Based on this equation and considering the fluorescence intensity distribution in the two-photon excitation volume, a theoretical expression for the PCH as a function of the number of molecules in the excitation volume is derived. For a single molecular species two parameters are sufficient to characterize the histogram completely, namely the average number of molecules within the observation volume and the detected photon counts per molecule per sampling time epsilon. The PCH for multiple molecular species, on the other hand, is generated by successively convoluting the photon counting distribution of each species with the others. The influence of the excitation profile upon the photon counting statistics for two relevant point spread functions (PSFs), the three-dimensional Gaussian PSF conventionally employed in confocal detection and the square of the Gaussian

  16. Identification of Atherosclerotic Plaques in Carotid Artery by Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Rocha, Rick; Villaverde, Antonio Balbin; Silveira, Landulfo; Costa, Maricília Silva; Alves, Leandro Procópio; Pasqualucci, Carlos Augusto; Brugnera, Aldo

    2008-04-01

    The aim of this work was to identify the presence of atherosclerotic plaques in carotid artery using the Fluorescence Spectroscopy. The most important pathogeny in the cardiovascular disorders is the atherosclerosis, which may affect even younger individuals. With approximately 1.2 million heart attacks and 750,000 strokes afflicting an aging American population each year, cardiovascular disease remains the number one cause of death. Carotid artery samples were obtained from the Autopsy Service at the University of São Paulo (São Paulo, SP, Brazil) taken from cadavers. After a histopathological analysis the 60 carotid artery samples were divided into two groups: normal (26) and atherosclerotic plaques (34). Samples were irradiated with the wavelength of 488 nm from an Argon laser. A 600 μm core optical fiber, coupled to the Argon laser, was used for excitation of the sample, whereas another 600 optical fiber, coupled to the spectrograph entrance slit, was used for collecting the fluorescence from the sample. Measurements were taken at different points on each sample and then averaged. Fluorescence spectra showed a single broad line centered at 549 nm. The fluorescence intensity for each sample was calculated by subtracting the intensity at the peak (550 nm) and at the bottom (510 nm) and then data were statistically analyzed, looking for differences between both groups of samples. ANOVA statistical test showed a significant difference (p<0,05) between both types of tissues, with regard to the fluorescence peak intensities. Our results indicate that this technique could be used to detect the presence of the atherosclerotic in carotid tissue.

  17. How to turn your pump–probe instrument into a multidimensional spectrometer: 2D IR and Vis spectroscopies via pulse shaping

    PubMed Central

    Shim, Sang-Hee; Zanni, Martin T.

    2010-01-01

    We have recently developed a new and simple way of collecting 2D infrared and visible spectra that utilizes a pulse shaper and a partly collinear beam geometry. 2D IR and Vis spectroscopies are powerful tools for studying molecular structures and their dynamics. They can be used to correlate vibrational or electronic eigenstates, measure energy transfer rates, and quantify the dynamics of lineshapes, for instance, all with femtosecond time-resolution. As a result, they are finding use in systems that exhibit fast dynamics, such as sub-millisecond chemical and biological dynamics, and in hard-to-study environments, such as in membranes. While powerful, these techniques have been difficult to implement because they require a series of femtosecond pulses to be spatially and temporally overlapped with precise time-resolution and interferometric phase stability. However, many of the difficulties associated with implementing 2D spectroscopies are eliminated by using a pulse shaper and a simple beam geometry, which substantially lowers the technical barriers required for researchers to enter this exciting field while simultaneously providing many new capabilities. The aim of this paper is to provide an overview of the methods for collecting 2D spectra so that an outsider considering using 2D spectroscopy in their own research can judge which approach would be most suitable for their research aims. This paper focuses primarily on 2D IR spectroscopy, but also includes our recent work on adapting this technology to collecting 2D Vis spectra. We review work that has already been published as well as cover several topics that we have not reported previously, including phase cycling methods to remove background signals, eliminate unwanted scatter, and shift data collection into the rotating frame. PMID:19290321

  18. Line scan fluorescence correlation spectroscopy for three-dimensional microfluidic flow velocity measurements

    NASA Astrophysics Data System (ADS)

    Pan, Xiaotao; Shi, Xianke; Korzh, Vladimir; Yu, Hanry; Wohland, Thorsten

    2009-03-01

    The flow direction of microfluidics in biological applications is not limited to two dimensions, but often extends to three dimensions. Currently there are optical methods available for the measurement of 3-D microfluidic flow vectors, but with low spatial resolution. Line scan fluorescence correlation spectroscopy (FCS) was proposed to determine flow directions in 2-D within microchannels and small blood vessels in our previous work. Importantly, its spatial resolution was demonstrated to be as good as 0.5 μm. In this work, we extend line scan FCS to the third dimension for the characterization of 3-D flow velocity vectors. The spatial resolution is close to the diffraction limit using a scan length of 0.5 μm in all three dimensions. The feasibility of line scan FCS for 3-D microfluidic flow is verified by measurements in microchannels and small blood vessels of zebrafish embryos.

  19. Fluorescence spectroscopy of polynuclear aromatic compounds in environmental monitoring.

    PubMed

    Kumke, M U; Löhmannsröben, H G; Roch, T

    1995-06-01

    The occurrence of polynuclear aromatic compounds (PAC) in the environment and experimental techniques suitable for the detection of PAC in environmental compartments are briefly reviewed. The specific requirements for on-site andin situ environmental analysis are outlined. Particular emphasis is given to fluorescence spectroscopic techniques for the investigation of humic acid- and soil-containing samples. Some examples of studies in the literature on Shpol'skii and jet spectroscopy and on laser-induced fluorescence (OF) measurements of PAC and mineral oils are highlighted. Contaminants in the environment are usually encountered as multicomponent mixtures in very complex matrices. Total fluorescence analysis in combination with the chemometrical technique of rank annihilation factor analysis (RAFA) was employed for the evaluation of a six-component PAC mixture in toluene. It was shown that even in the presence of strong spectral overlap the qualitative identification of all compounds and the reliable quantification of five substances was possible. Results are presented from our stationary and time-resolved fluorescence investigations of the interactions between pyrene and humic acid in water. The Stern-Volmer analysis showed a significant effect of pH on the static quenching efficiency which can be explained by the pH-dependent macromolecular structure of humic acids. Preliminary results from studies of the deactivation of triplet PAC and quenching of delayed fluorescence by humic acid are reported. LIF measurements of mineral oils directly from soil surfaces and of a model oil in a soil column were performed with a fiber-optic coupled multichannel spectrometer. The fluorescence intensity/ concentration relationships were established for a crude and a fuel oil; the corresponding lower limits of detection (LOD) were determined to be 0.025 and 0.125% m/m (mass/mass percentages). These detection limits are compared with realistic oil contaminations of soils. In a soil

  20. Classification of plum spirit drinks by synchronous fluorescence spectroscopy.

    PubMed

    Sádecká, J; Jakubíková, M; Májek, P; Kleinová, A

    2016-04-01

    Synchronous fluorescence spectroscopy was used in combination with principal component analysis (PCA) and linear discriminant analysis (LDA) for the differentiation of plum spirits according to their geographical origin. A total of 14 Czech, 12 Hungarian and 18 Slovak plum spirit samples were used. The samples were divided in two categories: colorless (22 samples) and colored (22 samples). Synchronous fluorescence spectra (SFS) obtained at a wavelength difference of 60 nm provided the best results. Considering the PCA-LDA applied to the SFS of all samples, Czech, Hungarian and Slovak colorless samples were properly classified in both the calibration and prediction sets. 100% of correct classification was also obtained for Czech and Hungarian colored samples. However, one group of Slovak colored samples was classified as belonging to the Hungarian group in the calibration set. Thus, the total correct classifications obtained were 94% and 100% for the calibration and prediction steps, respectively. The results were compared with those obtained using near-infrared (NIR) spectroscopy. Applying PCA-LDA to NIR spectra (5500-6000 cm(-1)), the total correct classifications were 91% and 92% for the calibration and prediction steps, respectively, which were slightly lower than those obtained using SFS.

  1. Fluorescence correlation spectroscopy of repulsive systems: theory, simulation, and experiment.

    PubMed

    Feng, Ligang; Yang, Jingfa; Zhao, Jiang; Wang, Dapeng; Koynov, Kaloian; Butt, Hans-Jürgen

    2013-06-07

    The theoretical basis of fluorescence correlation spectroscopy (FCS) for repulsive systems, such as charged colloids or macromolecules, has been further expanded and developed. It is established that the collective correlation function can no longer be fitted using the theoretical model of non-interacting systems. Also, it is discovered that the collective correlation function can be divided into two parts: a self-part and a distinct-part, named as the self-correlation and cross-correlation function, respectively. The former indicates the self-diffusion of objects, while the latter describes mutual interactions. Dual-color fluorescence cross-correlation spectroscopy provides the direct measurements of the two parts. The particle concentration and mean squared displacement of single particles can be deduced from the self-correlation function, while the correlation volume between particles can be approximated from the cross-correlation function. In the case of charged colloids, the Debye length of the solution and particle surface charge number can be fitted from the cross-correlation function. These theoretical results are successfully proven using Brownian dynamics simulations and preliminary FCS experiments for model charged colloidal systems.

  2. A universal model of restricted diffusion for fluorescence correlation spectroscopy.

    PubMed

    Piskorz, Tomasz K; Ochab-Marcinek, Anna

    2014-05-08

    Fluorescence correlation spectroscopy (FCS) is frequently used to study the processes of restricted diffusion. The most important quantity to determine is the size of the structures that hinder the Brownian motion of the molecules. We study three qualitatively different models of restricted diffusion, widely applied in biophysics and material science: Diffusion constrained by elastic force (i), walking confined diffusion (ii), and hop diffusion (iii). They cover the diversity of statistical behaviors, from purely Gaussian (i) to sharply non-Gaussian on intermediate time scales (ii) and, additionally, discrete (iii). We test whether one can use the Gaussian approximation of the FCS autocorrelation function to interpret the non-Gaussian data. We show that (i-iii) have approximately the same mean square displacements. Using simulations, we show that the FCS data suspected of restricted diffusion can be reliably interpreted using one archetypal model (i). Even if the underlying mechanism of the restriction is different or unknown, the accuracy of fitting the confinement size is excellent, and diffusion coefficients are also estimated with a good accuracy. This study gives a physical insight into the statistical behavior of different types of restricted diffusion and into the ability of fluorescence correlation spectroscopy to distinguish between them.

  3. Fluorescence Spectroscopy and Chemometric Modeling for Bioprocess Monitoring

    PubMed Central

    Faassen, Saskia M.; Hitzmann, Bernd

    2015-01-01

    On-line sensors for the detection of crucial process parameters are desirable for the monitoring, control and automation of processes in the biotechnology, food and pharma industry. Fluorescence spectroscopy as a highly developed and non-invasive technique that enables the on-line measurements of substrate and product concentrations or the identification of characteristic process states. During a cultivation process significant changes occur in the fluorescence spectra. By means of chemometric modeling, prediction models can be calculated and applied for process supervision and control to provide increased quality and the productivity of bioprocesses. A range of applications for different microorganisms and analytes has been proposed during the last years. This contribution provides an overview of different analysis methods for the measured fluorescence spectra and the model-building chemometric methods used for various microbial cultivations. Most of these processes are observed using the BioView® Sensor, thanks to its robustness and insensitivity to adverse process conditions. Beyond that, the PLS-method is the most frequently used chemometric method for the calculation of process models and prediction of process variables. PMID:25942644

  4. Laser-induced fluorescence and dispersed fluorescence spectroscopy of jet-cooled 1-phenylpropargyl radical

    NASA Astrophysics Data System (ADS)

    Reilly, Neil J.; Nakajima, Masakazu; Gibson, Bligh A.; Schmidt, Timothy W.; Kable, Scott H.

    2009-04-01

    The D1(A2″)-D0(A2″) electronic transition of the resonance-stabilized 1-phenylpropargyl radicalooled discharge of 3-phenyl-1-propyne, has been investigated in detail by laser-induced fluorescence excitation and dispersed single vibronic level fluorescence (SVLF) spectroscopy. The transition is dominated by the origin band at 21 007 cm-1, with weaker Franck-Condon activity observed in a' fundamentals and even overtones and combinations of a″ symmetry. Ab initio and density functional theory calculations of the D0 and D1 geometries and frequencies were performed to support and guide the experimental assignments throughout. Analysis of SVLF spectra from 16 D1 vibronic levels has led to the assignment of 15 fundamental frequencies in the excited state and 19 fundamental frequencies in the ground state; assignments for many more normal modes not probed directly by fluorescence spectroscopy are also suggested. Duschinsky mixing, in which the excited state normal modes are rotated with respect to the ground state modes, is prevalent throughout, in vibrations of both a' and a″ symmetry.

  5. Comparison of absorption, fluorescence, and polarization spectroscopy of atomic rubidium

    NASA Astrophysics Data System (ADS)

    Ashman, Seth; Stifler, Cayla; Romero, Joaquin

    2015-05-01

    An ongoing spectroscopic investigation of atomic rubidium utilizes a two-photon, single-laser excitation process. Transitions accessible with our tunable laser include 5P1 / 2F' <-- 5S1 / 2 F and 5P3 / 2F' <-- 5S1 / 2 F . The laser is split into a pump and probe beam to allow for Doppler-free measurements of transitions between hyperfine levels. The pump and probe beams are overlapped in a counter-propagating geometry and the laser frequency scans over a transition. Absorption, fluorescence and polarization spectroscopy techniques are applied to this basic experimental setup. The temperature of the vapor cell and the power of the pump and probe beams have been varied to explore line broadening effects and signal-to-noise of each technique. This humble setup will hopefully grow into a more robust experimental arrangement in which double resonance, two-laser excitations are used to explore hyperfine state changing collisions between rubidium atoms and noble gas atoms. Rb-noble gas collisions can transfer population between hyperfine levels, such as 5P3 / 2 (F' = 3) <-- Collision 5P3 / 2 (F ' = 2) , and the probe beam couples 7S1 / 2 (F'' = 2) <-- 5P3 / 2 (F' = 3) . Polarization spectroscopy signal depends on the rate of population transfer due to the collision as well as maintaining the orientation created by the pump laser. Fluorescence spectroscopy relies only on transfer of population due to the collision. Comparison of these techniques yields information regarding the change of the magnetic sublevels, mF, during hyperfine state changing collisions.

  6. Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

    PubMed Central

    Guan, Yinghua; Meurer, Matthias; Raghavan, Sarada; Rebane, Aleksander; Lindquist, Jake R.; Santos, Sofia; Kats, Ilia; Davidson, Michael W.; Mazitschek, Ralph; Hughes, Thomas E.; Drobizhev, Mikhail; Knop, Michael; Shah, Jagesh V.

    2015-01-01

    We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells. PMID:25877871

  7. 2D/3D cryo x-ray fluorescence imaging at the bionanoprobe at the advanced photon source

    SciTech Connect

    Chen, S. Vine, D. J.; Lai, B.; Paunesku, T.; Yuan, Y.; Woloschak, G. E.; Deng, J.; Jin, Q.; Hong, Y. P.; Flachenecker, C.; Hornberger, B.; Brister, K.; Jacobsen, C.; Vogt, S.

    2016-01-28

    Trace elements, particularly metals, play very important roles in biological systems. Synchrotron-based hard X-ray fluorescence microscopy offers the most suitable capabilities to quantitatively study trace metals in thick biological samples, such as whole cells and tissues. In this manuscript, we have demonstrated X-ray fluorescence imaging of frozen-hydrated whole cells using the recent developed Bionanoprobe (BNP). The BNP provides spatial resolution down to 30 nm and cryogenic capabilities. Frozen-hydrated biological cells have been directly examined on a sub-cellular level at liquid nitrogen temperatures with minimal sample preparation.

  8. Mean cell size and collagen orientation from 2D Fourier analysis on confocal laser scanning microscopy and two-photon fluorescence microscopy on human skin in vivo

    NASA Astrophysics Data System (ADS)

    Lucassen, Gerald W.; Bakker, Bernard L.; Neerken, Sieglinde; Hendriks, Rob F. M.

    2003-07-01

    We present results from 2D Fourier analysis on 3D stacks of images obtained by confocal laser scanning reflectance microscopy (CLSM) and two-photon fluorescence microscopy (2PM) on human skin in vivo. CLSM images were obtained with a modified commercial system (Vivascope1000, Lucid Inc, excitation wavelength 830 nm) equipped with a piezo-focusing element (350 μm range) for depth positioning of the objective lens. 2PM was performed with a specially designed set-up with excitation wavelength 730 nm. Mean cell size in the epidermal layer and structural orientation in the dermal layer have been determined as a function of depth by 2D Fourier analysis. Fourier analysis on microscopic images enables automatic non-invasive quantitative structural analysis (mean cell size and orientation) of living human skin.

  9. Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Smith, Elizabeth Myhra

    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

  10. Monitoring helicase-catalyzed DNA unwinding by fluorescence anisotropy and fluorescence cross-correlation spectroscopy.

    PubMed

    Xi, Xu Guang; Deprez, Eric

    2010-07-01

    In order to elucidate molecular mechanism of helicases, we have developed two new rapid and sensitive fluorescence assays to measure helicase-mediated DNA unwinding. The fluorescence anisotropy (FA) assay takes the advantage of the substantial change in fluorescence polarization upon helicase binding to DNA and DNA unwinding. The extent of depolarization depends on the rate of tumbling of the fluorescently labeled DNA molecule, which decreases with increasing size. This assay therefore can simultaneously monitor the DNA binding of helicase and the subsequent helicase-catalyzed DNA unwinding in real-time. For size limitation reasons, the FA approach is more suitable for single-turnover kinetic studies. A fluorescence cross-correlation spectroscopy method (FCCS) is also described for measuring DNA unwinding. This assay is based on the degree of concomitant diffusion of the two complementary DNA strands in a small excitation volume, each labeled by a different color. The decrease in the amplitude of the cross-correlation signal is then directly related to the unwinding activity. By contrast with FA, the FCCS-based assay can be used to measure the unwinding activity under both single- and multiple-turnover conditions, with no limitation related to the size of the DNA strands constituting the DNA substrate. These methods used together have proven to be useful for studying molecular mechanism underlying efficient motor function of helicases. Here, we describe the theoretical basis and framework of FA and FCCS and some practical implications for measuring DNA binding and unwinding. We discuss sample preparation and potential troubleshooting. Special attention is paid to instrumentation, data acquisition and analysis.

  11. Study on antibacterial alginate-stabilized copper nanoparticles by FT-IR and 2D-IR correlation spectroscopy

    PubMed Central

    Díaz-Visurraga, Judith; Daza, Carla; Pozo, Claudio; Becerra, Abraham; von Plessing, Carlos; García, Apolinaria

    2012-01-01

    Background The objective of this study was to clarify the intermolecular interaction between antibacterial copper nanoparticles (Cu NPs) and sodium alginate (NaAlg) by Fourier transform infrared spectroscopy (FT-IR) and to process the spectra applying two-dimensional infrared (2D-IR) correlation analysis. To our knowledge, the addition of NaAlg as a stabilizer of copper nanoparticles has not been previously reported. It is expected that the obtained results will provide valuable additional information on: (1) the influence of reducing agent ratio on the formation of copper nanoparticles in order to design functional nanomaterials with increased antibacterial activity, and (2) structural changes related to the incorporation of Cu NPs into the polymer matrix. Methods Cu NPs were prepared by microwave heating using ascorbic acid as reducing agent and NaAlg as stabilizing agent. The characterization of synthesized Cu NPs by ultraviolet visible spectroscopy, transmission electron microscopy (TEM), electron diffraction analysis, X-ray diffraction (XRD), and semiquantitative analysis of the weight percentage composition indicated that the average particle sizes of Cu NPs are about 3–10 nm, they are spherical in shape, and consist of zerovalent Cu and Cu2O. Also, crystallite size and relative particle size of stabilized Cu NPs were calculated by XRD using Scherrer’s formula and FT from the X-ray diffraction data. Thermogravimetric analysis, differential thermal analysis, differential scanning calorimetry (DSC), FT-IR, second-derivative spectra, and 2D-IR correlation analysis were applied to studying the stabilization mechanism of Cu NPs by NaAlg molecules. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of stabilized Cu NPs against five bacterial strains (Staphylococccus aureus ATCC 6538P, Escherichia coli ATCC 25922 and O157: H7, and Salmonella enterica serovar Typhimurium ATCC 13311 and 14028) were evaluated with macrodilution

  12. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    SciTech Connect

    Laurence, Ted A.; Ly, Sonny; Bourguet, Feliza; Fischer, Nicholas O.; Coleman, Matthew A.

    2014-08-14

    Fluorescence correlation spectroscopy (FCS) is an important technique for studying biochemical interactions dynamically that may be used in vitro and in cell-based studies. It is generally claimed that FCS may only be used at nM concentrations. We show that this general consensus is incorrect and that the limitation to nM concentrations is not fundamental but due to detector limits as well as laser fluctuations. With a high count rate detector system and applying laser fluctuation corrections, we demonstrate FCS measurements up to 38 μM with the same signal-to-noise as at lower concentrations. Optical nanoconfinement approaches previously used to increase the concentration range of FCS are not necessary, and further increases above 38 μM may be expected using detectors and detector arrays with higher saturation rates and better laser fluctuation corrections. This approach greatly widens the possibilities of dynamic measurements of biochemical interactions using FCS at physiological concentrations.

  13. Detecting Nanodomains in Living Cell Membrane by Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    He, Hai-Tao; Marguet, Didier

    2011-05-01

    Cell membranes actively participate in numerous cellular functions. Inasmuch as bioactivities of cell membranes are known to depend crucially on their lateral organization, much effort has been focused on deciphering this organization on different length scales. Within this context, the concept of lipid rafts has been intensively discussed over recent years. In line with its ability to measure diffusion parameters with great precision, fluorescence correlation spectroscopy (FCS) measurements have been made in association with innovative experimental strategies to monitor modes of molecular lateral diffusion within the plasma membrane of living cells. These investigations have allowed significant progress in the characterization of the cell membrane lateral organization at the suboptical level and have provided compelling evidence for the in vivo existence of raft nanodomains. We review these FCS-based studies and the characteristic structural features of raft nanodomains. We also discuss the findings in regards to the current view of lipid rafts as a general membrane-organizing principle.

  14. Parameter estimation and analysis model selections in fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Dong, Shiqing; Zhou, Jie; Ding, Xuemei; Wang, Yuhua; Xie, Shusen; Yang, Hongqin

    2016-10-01

    Fluorescence correlation spectroscopy (FCS) is a powerful technique that could provide high temporal resolution and detection for the diffusions of biomolecules at extremely low concentrations. The accuracy of this approach primarily depends on experimental condition requirements and the data analysis model. In this study, we have set up a confocal-based FCS system. And then we used a Rhodamine6G solution to calibrate the system and get the related parameters. An experimental measurement was carried out on one-component solution to evaluate the relationship between a certain number of molecules and concentrations. The results showed FCS system we built was stable and valid. Finally, a two-component solution experiment was carried out to show the importance of analysis model selection. It is a promising method for single molecular diffusion study in living cells.

  15. Fluorescence correlation spectroscopy evidence for structural heterogeneity in ionic liquids

    SciTech Connect

    Guo, J C; Baker, G. A.; Hillesheim, P. C.; Dai, S.; Shaw, R. W.; Mahurin, S., M.

    2011-01-01

    In this work, we provide new experimental evidence for chain length-dependent self-aggregation in room temperature ionic liquids (RTILs) using fluorescence correlation spectroscopy (FCS). In studying a homologous series of N-alkyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl) imide, [C{sub n}MPy][Tf{sub 2}N] RTILs of varying alkyl chain length (n = 3, 4, 6, 8, and 10), biphasic rhodamine 6G solute diffusion dynamics were observed; both the fast and slow diffusion coefficients decreased with increasing alkyl chain length, with the relative contribution from slower diffusion increasing for longer-chain [C{sub n}MPy][Tf{sub 2}N]. We propose that the biphasic diffusion dynamics originate from self-aggregation of the nonpolar alkyl chains in the cationic [CnMPy]{sup +}.

  16. Fluorescence and UV-vis Spectroscopy of Synovial Fluids

    NASA Astrophysics Data System (ADS)

    Pinti, Marie J.; Stojilovic, Nenad; Kovacik, Mark W.

    2009-10-01

    Total joint arthroplasty involves replacing the worn cartilaginous surfaces of the joint with man-made materials that are designed to be biocompatible and to withstand mechanical stresses. Commonly these bearing materials consist of metallic alloys (TiAlV or CoCrMo) and UHMWPE. Following joint arthroplasty, the normal generation of micro-metallic wear debris particles that dislodge from the prosthesis has been shown to cause inflammatory aseptic osteolysis (bone loss) that ultimately results in the failure of the implant. Here we report our results on the novel use of Fluorescence and UV-vis spectroscopy to investigate the metallic content of synovial fluid specimens taken from postoperative total knee arthroplasties. Preliminary finding showed presence of alumina and chromium is some specimens. The ability to detect and monitor the wear rate of these implants could have far reaching implications in the prevention of metallic wear-debris induced osteolysis and impending implant failure.

  17. Quick tour of fluorescence correlation spectroscopy from its inception.

    PubMed

    Elson, Elliot L

    2004-01-01

    Fluorescence correlation spectroscopy (FCS) was originally developed in the early 1970s as a way to measure the kinetics of chemical reactions under zero perturbation conditions. At its inception, the measurement was difficult due to experimental limitations and was primarily used during the 1970s and 1980s to characterize diffusion. More recently, as a result of technological advances, FCS measurements have become easier and more versatile. In addition to measurements of diffusion both in solution and in cells, FCS is now also used to measure not only chemical reaction kinetics but also extents of molecular aggregation, the dynamics of photophysical processes, conformational fluctuations, molecular interactions in solution and in cells, and has even found application as a pharmaceutical screening method. From its inception to the present, the contributions of Webb and his coworkers have had a central and defining role in the development and applications of FCS.

  18. Characterization of binding site heterogeneity for copper within dissolved organic matter fractions using two-dimensional correlation fluorescence spectroscopy.

    PubMed

    Hur, Jin; Lee, Bo-Mi

    2011-06-01

    The heterogeneity of copper binding characteristics for dissolved organic matter (DOM) fractions was investigated based on the fluorescence quenching of the synchronous fluorescence spectra upon the addition of copper and two-dimensional correlation spectroscopy (2D-COS). Hydrophobic acid (HoA) and hydrophilic (Hi) fractions of two different DOM (algal and leaf litter DOM) were used for this study. For both DOM, fluorescence quenching occurred at a wider range of wavelengths for the HoA fractions compared to the Hi fractions. The combined information of the synchronous and asynchronous maps derived from 2D-COS provided a clear picture of the heterogeneous distribution of the copper binding sites within each DOM fraction, which was not readily recognized by a simple comparison of the changes in the synchronous fluorescence spectra upon the addition of copper. For the algal DOM, higher stability constants were exhibited for the HoA versus the Hi fractions. The logarithms of the stability constants ranged from 4.8 to 6.1 and from 4.5 to 5.0 for the HoA and the Hi fractions of the algal DOM, respectively, depending on the associated wavelength and the fitted models. In contrast, no distinctive difference in the binding characteristics was found between the two fractions of the leaf litter DOM. This suggests that influences of the structural and chemical properties of DOM on copper binding may differ for DOM from different sources. The relative difference of the calculated stability constants within the DOM fractions were consistent with the sequential orders interpreted from the asynchronous 2D-COS. It is expected that 2D-COS will be widely applied to other DOM studies requiring detailed information on the heterogeneous nature and subsequent effects under a range of environmental conditions.

  19. [Outlier Detection of Time Series Three-Dimensional Fluorescence Spectroscopy].

    PubMed

    Yu, Shao-hui; Zhang, Yu-jun; Zhao, Nan-jing

    2015-06-01

    The qualitative and quantitative analysis are often interfered by the outliers in time series three-dimensional fluorescence spectroscopy. In this work, an efficient outlier detection method is proposed by taking advantage of the characteristics in time dimension and the spectral dimension. Firstly, the wavelength points that are mostly the outliers are extracted by the variance in time dimension. Secondly, by the analysis of the existence styles of outliers and similarity score of any two samples, the cumulative similarity is introduced in spectral dimension. At last, fluorescence intensity at each wavelength of all samples is modified by the correction matrix in time dimension and the outlier detection is completed according the to cumulative similarity scores. The application of the correction matrix in time dimension not only improves the validity of the method but also reduces the computation by the choice of characteristics region in correction matrix. Numerical experiments show that the outliers can still be detected by the 50 percent of all points in spectral dimension.

  20. Evaluation of actinic cheilitis using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Pratavieira, Sebastião.; Takahama, Ademar; Souza Azevedo, Rebeca; Kurachi, Cristina

    2016-03-01

    Actinic cheilitis is a potentially malignant disorder that mostly affects the vermilion border of the lower lip and can lead to squamous cell carcinoma. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. Late diagnosis is a limiting factor of therapeutic possibilities available to treat oral cancer. The diagnosis of actinic cheilitis is mainly based on clinical and histopathological analysis and it is a time consuming procedure to get the results. Information about the organization and chemical composition of the tissues can be obtained using fluorescence lifetime spectroscopy techniques without the need for biopsy. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and allow a quick and non-invasive clinical investigation of injuries and to help clinicians with the early diagnosis of actinic cheilitis. This study aims to evaluate the fluorescence lifetime parameters at the discrimination of three degrees of epithelial dysplasia, the most important predictor of malignant development, described in up to 100% of actinic cheilitis cases.

  1. Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Liang, Lifang; Xing, Da; Chen, Tongshen; Pei, Yihui

    2007-11-01

    Fluorescence correlation spectroscopy (FCS) is a new kind of real-time, high-speed and single-molecule technique. It is used to detect the kinetic characteristics of fluorescent dye such as diffusion coefficient in the aqueous solution. Combined with confocal microscope optics, it has been now widely applied in cell biological research. Through a time correlation analysis of spontaneous intensity fluctuations, this technique with EGFP as a probe is capable of determining viscosity of fluids according to Stokes-Einstein equation. Nucleoplasmic viscosity is an important physical parameter to quantify the rheological characteristics of the nucleoplasm. Investigation on nucleoplasmic viscosity plays an important role in further understanding intranuclear environment. In this paper, FCS is introduced to noninvasively investigate nucleoplasmic viscosity of living cells. The results show that nucleoplasmic viscosity of lung adenocarcinoma (ASTC-a-1) cells is 2.55+/-0.61 cP and nucleoplasmic viscosity is larger than cytoplasmic viscosity at 37 °C (pH 7.4). In addition, significant changes in nucleoplasmic viscosity are detected by FCS when cells are exposed to hyper or hypotonic medium. Our study suggests that FCS can be used to detect the kinetic characteristics of biomolecules in living cells and thus helps to investigate the dynamic changes of the microenvironment in the cell.

  2. Characterization of Titratable Amphiphiles in Lipid Membranes by Fluorescence Spectroscopy.

    PubMed

    Pierrat, Philippe; Lebeau, Luc

    2015-11-17

    Understanding the ionization behavior of lipid membranes is a key parameter for successful development of lipid-based drug delivery systems. Accurate determination of the ionization state of a titratable species incorporated in a lipid bilayer however requires special care. Herein we investigated the behavior of titratable lipids in liposomes by fluorescence spectroscopy and determined which extrinsic parameters-i.e., besides those directly related to their molecular structure-determine their ionization state. Two fluorescent dyes, TNS and R18, have been used to investigate basic and acidic titratable lipids, respectively. Our results suggest that the titration behavior of the ionizable lipid in the membrane is more sensitive to the composition of the membrane and to its physical state than to the presence of solutes in the aqueous phase. Essentially overlooked in earlier studies on ionizable lipid assemblies, the concentration of the titratable lipid in the membrane was found to have a major effect on the ionization state of the lipid polar head. This may result in a shift in the apparent pKa value which may be as large as two pKa units and cannot be satisfactorily predicted.

  3. Optical biopsy fiber-based fluorescence spectroscopy instrumentation

    NASA Astrophysics Data System (ADS)

    Katz, Alvin; Ganesan, Singaravelu; Yang, Yuanlong; Tang, Gui C.; Budansky, Yury; Celmer, Edward J.; Savage, Howard E.; Schantz, Stimson P.; Alfano, Robert R.

    1996-04-01

    Native fluorescence spectroscopy of biomolecules has emerged as a new modality to the medical community in characterizing the various physiological conditions of tissues. In the past several years, many groups have been working to introduce the spectroscopic methods to diagnose cancer. Researchers have successfully used native fluorescence to distinguish cancerous from normal tissue samples in rat and human tissue. We have developed three generations of instruments, called the CD-scan, CD-ratiometer and CD-map, to allow the medical community to use optics for diagnosing tissue. Using ultraviolet excitation and emission spectral measurements on both normal and cancerous tissue of the breast, gynecology, colon, and aerodigestive tract can be separated. For example, from emission intensities at 340 nm to 440 nm (300 nm excitation), a statistically consistent difference between malignant tissue and normal or benign tissue is observed. In order to utilize optical biopsy techniques in a clinical setting, the CD-scan instrument was developed, which allows for rapid and reliable in-vitro and in-vivo florescence measurements of the aerodigestive tract with high accuracy. The instrumentation employs high sensitivity detection techniques which allows for lamp excitation, small diameter optical fiber probes; the higher spatial resolution afforded by the small diameter probes can increase the ability to detect smaller tumors. The fiber optic probes allow for usage in the aerodigestive tract, cervix and colon. Needle based fiber probes have been developed for in-vivo detection of breast cancer.

  4. [Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy].

    PubMed

    Liang, Li-Fang; Da, Xing; Chen, Tong-Sheng; Pei, Yi-Hui

    2009-02-01

    In order to non-invasively investigate nucleoplasmic viscosity in real time with good temporal resolution, the present study firstly introduced a new method based on fluorescence correlation spectroscopy (FCS). FCS is a kind of single-molecule technique with high temporal and spatial resolution to analyze the dynamics of fluorescent molecules in nanomolar concentration. Through a time correlation analysis of spontaneous intensity fluctuations, this technique in conjunction with EGFP as a probe is capable of determining nucleoplasmic viscosity in terms of Stokes-Einstein equation as well as its corresponding analysis of the diffusion coefficient for EGFP in the nucleus. The results showed that nucleoplasmic viscosity of ASTC-a-1 cells and HeLa cells were respectively (2.55 +/- 0.61) cP and (2.04 +/- 0.49) cP at pH 7.4 and 37 degrees C, consistent with the results by traditional methods, and nucleoplasmic viscosity was found to be larger than cytoplasmic viscosity. Meanwhile, the real-time analysis of nucleoplasmic viscosity in living cells exposed to hypotonic media proved that FCS could be used to track the changing rheological characteristics of the nucleoplasm in living cells. Taken together, this study suggests that FCS provides an accurate and non-invasive method to investigate the microenvironment in living cells on the femtoliter scale and it can be used as a powerful tool in researches on the dynamical processes of intracellular molecules.

  5. Single-Molecule Fluorescence Spectroscopy of Photosynthetic Systems.

    PubMed

    Kondo, Toru; Chen, Wei Jia; Schlau-Cohen, Gabriela S

    2017-01-25

    Photosynthesis begins when a network of pigment-protein complexes captures solar energy and transports it to the reaction center, where charge separation occurs. When necessary (under low light conditions), photosynthetic organisms perform this energy transport and charge separation with near unity quantum efficiency. Remarkably, this high efficiency is maintained under physiological conditions, which include thermal fluctuations of the pigment-protein complexes and changing local environments. These conditions introduce multiple types of heterogeneity in the pigment-protein complexes, including structural heterogeneity, energetic heterogeneity, and functional heterogeneity. Understanding how photosynthetic light-harvesting functions in the face of these fluctuations requires understanding this heterogeneity, which, in turn, requires characterization of individual pigment-protein complexes. Single-molecule spectroscopy has the power to probe individual complexes. In this review, we present an overview of the common techniques for single-molecule fluorescence spectroscopy applied to photosynthetic systems and describe selected experiments on these systems. We discuss how these experiments provide a new understanding of the impact of heterogeneity on light harvesting and thus how these systems are optimized to capture sunlight under physiological conditions.

  6. Advanced Photoemission Spectroscopy Investigations Correlated with DFT Calculations on the Self-Assembly of 2D Metal Organic Frameworks Nano Thin Films.

    PubMed

    Elzein, Radwan; Chang, Chun-Min; Ponomareva, Inna; Gao, Wen-Yang; Ma, Shengqian; Schlaf, Rudy

    2016-11-16

    Metal-organic frameworks (MOFs) deposited from solution have the potential to form 2-dimensional supramolecular thin films suitable for molecular electronic applications. However, the main challenges lie in achieving selective attachment to the substrate surface, and the integration of organic conductive ligands into the MOF structure to achieve conductivity. The presented results demonstrate that photoemission spectroscopy combined with preparation in a system-attached glovebox can be used to characterize the electronic structure of such systems. The presented results demonstrate that porphyrin-based 2D MOF structures can be produced and that they exhibit similar electronic structure to that of corresponding conventional porphyrin thin films. Porphyrin MOF multilayer thin films were grown on Au substrates prefunctionalized with 4-mercaptopyridine (MP) via incubation in a glovebox, which was connected to an ultrahigh vacuum system outfitted with photoelectron spectroscopy. The thin film growth process was carried out in several sequential steps. In between individual steps the surface was characterized by photoemission spectroscopy to determine the valence bands and evaluate the growth mode of the film. A comprehensive evaluation of X-ray photoemission spectroscopy (XPS), ultraviolet photoelectron spectroscopy (UPS), and inverse photoemission spectroscopy (IPES) data was performed and correlated with density functional theory (DFT) calculations of the density of states (DOS) of the films involved to yield the molecular-level insights into the growth and the electronic properties of MOF-based 2D thin films.

  7. Fluorescence and Diffuse Reflectance Spectroscopy for Breast Cancer Diagnosis During Core Needle Biopsy

    DTIC Science & Technology

    2007-09-01

    The goal of this project is to explore the potential of using tissue fluorescence and diffuse reflectance spectroscopy for breast cancer detection...sensor based on tissue fluorescence and diffuse reflectance spectroscopy as an adjunct diagnostic tool, which has the potential to provide guidance for core needle breast biopsy.

  8. Methods of single-molecule fluorescence spectroscopy and microscopy

    NASA Astrophysics Data System (ADS)

    Moerner, W. E.; Fromm, David P.

    2003-08-01

    Optical spectroscopy at the ultimate limit of a single molecule has grown over the past dozen years into a powerful technique for exploring the individual nanoscale behavior of molecules in complex local environments. Observing a single molecule removes the usual ensemble average, allowing the exploration of hidden heterogeneity in complex condensed phases as well as direct observation of dynamical state changes arising from photophysics and photochemistry, without synchronization. This article reviews the experimental techniques of single-molecule fluorescence spectroscopy and microscopy with emphasis on studies at room temperature where the same single molecule is studied for an extended period. Key to successful single-molecule detection is the need to optimize signal-to-noise ratio, and the physical parameters affecting both signal and noise are described in detail. Four successful microscopic methods including the wide-field techniques of epifluorescence and total internal reflection, as well as confocal and near-field optical scanning microscopies are described. In order to extract the maximum amount of information from an experiment, a wide array of properties of the emission can be recorded, such as polarization, spectrum, degree of energy transfer, and spatial position. Whatever variable is measured, the time dependence of the parameter can yield information about excited state lifetimes, photochemistry, local environmental fluctuations, enzymatic activity, quantum optics, and many other dynamical effects. Due to the breadth of applications now appearing, single-molecule spectroscopy and microscopy may be viewed as useful new tools for the study of dynamics in complex systems, especially where ensemble averaging or lack of synchronization may obscure the details of the process under study.

  9. 2D ion velocity distribution function measurements by laser-induced fluorescence above a radio-frequency biased silicon wafer

    NASA Astrophysics Data System (ADS)

    Moore, Nathaniel; Gekelman, Walter; Pribyl, Patrick; Zhang, Yiting; Kushner, Mark

    2012-10-01

    Ion dynamics have been measured in the sheath above a 30 cm diameter, 2.2 MHz-biased silicon wafer in a plasma processing etch tool using laser-induced fluorescence (LIF). The velocity distribution function of argon ions was measured at thousands of positions above and radially along the edge of the wafer by sending a planar laser sheet from a pulsed, tunable dye laser into the tool. The RF sheath is clearly resolved. The laser sheet entered the machine both parallel and perpendicular to the wafer in order to measure the distribution function for both parallel and perpendicular velocities/energies (0.4 eV < Emax<600 eV). The resulting fluorescence was recorded using a fast CCD camera, which provided spatial (0.4 mm) and temporal (30 ns) resolution. Data was taken at eight different phases of the 2.2 MHz cycle. The distribution functions were found to be spatially non-uniform near the edge of the wafer and the distribution of energies extremely phase-dependent. Several cm above the wafer the distribution is Maxwellian and independent of phase. Results are compared with simulations; for example, the experimental time-averaged ion energy distribution function compares favorably with a computer model carefully constructed to emulate the device.

  10. 2D ion velocity distribution function measurements by laser-induced fluorescence above a radio-frequency biased silicon wafer

    NASA Astrophysics Data System (ADS)

    Moore, Nathaniel; Gekelman, Walter; Pribyl, Patrick; Zhang, Yiting; Kushner, Mark

    2012-10-01

    Ion dynamics have been measured in the sheath above a 30 cm diameter, 2.2 MHz-biased silicon wafer in a plasma processing etch tool using laser-induced fluorescence (LIF). The velocity distribution function of argon ions was measured at thousands of positions above and radially along the edge of the wafer by sending a planar laser sheet from a pulsed, tunable dye laser into the tool. The RF sheath is clearly resolved. The laser sheet entered the machine both parallel and perpendicular to the wafer in order to measure the distribution function for both parallel and perpendicular velocities/energies (0.4 eV < Emax< 600 eV). The resulting fluorescence was recorded using a fast CCD camera, which provided spatial (0.4 mm) and temporal (30 ns) resolution. Data was taken at eight different phases of the 2.2 MHz cycle. The distribution functions were found to be spatially non-uniform near the edge of the wafer and the distribution of energies extremely phase-dependent. Several cm above the wafer the distribution is Maxwellian and independent of phase. Results are compared with simulations; for example, the experimental time-averaged ion energy distribution function compares favorably with a computer model carefully constructed to emulate the device.

  11. Residue-Specific Structural Kinetics of Proteins through the Union of Isotope Labeling, Mid-IR Pulse Shaping, and Coherent 2D IR Spectroscopy

    PubMed Central

    Middleton, Chris T.; Woys, Ann Marie; Mukherjee, Sudipta S.; Zanni, Martin T.

    2010-01-01

    We describe a methodology for studying protein kinetics using a rapid-scan technology for collecting 2D IR spectra. In conjunction with isotope labeling, 2D IR spectroscopy is able to probe the secondary structure and environment of individual residues in polypeptides and proteins. It is particularly useful for membrane and aggregate proteins. Our rapid-scan technology relies on a mid-IR pulse shaper that computer generates the pulse shapes, much like in an NMR spectrometer. With this device, data collection is faster, easier, and more accurate. We describe our 2D IR spectrometer, as well as protocols for 13C=18O isotope labeling, and then illustrate the technique with an application to the aggregation of the human islet amyloid polypeptide form type 2 diabetes. PMID:20472067

  12. Discrimination of adulterated milk based on two-dimensional correlation spectroscopy (2D-COS) combined with kernel orthogonal projection to latent structure (K-OPLS).

    PubMed

    Yang, Renjie; Liu, Rong; Xu, Kexin; Yang, Yanrong

    2013-12-01

    A new method for discrimination analysis of adulterated milk and pure milk is proposed by combining two-dimensional correlation spectroscopy (2D-COS) with kernel orthogonal projection to latent structure (K-OPLS). Three adulteration types of milk with urea, melamine, and glucose were prepared, respectively. The synchronous 2D spectra of adulterated milk and pure milk samples were calculated. Based on the characteristics of 2D correlation spectra of adulterated milk and pure milk, a discriminant model of urea-tainted milk, melamine-tainted milk, glucose-tainted milk, and pure milk was built by K-OPLS. The classification accuracy rates of unknown samples were 85.7, 92.3, 100, and 87.5%, respectively. The results show that this method has great potential in the rapid discrimination analysis of adulterated milk and pure milk.

  13. Interactions of gemini surfactants with two model proteins: NMR, CD, and fluorescence spectroscopies.

    PubMed

    Amiri, Razieh; Bordbar, Abdol-Khalegh; García-Mayoral, Ma Flor; Khosropour, Ahmad Reza; Mohammadpoor-Baltork, Iraj; Menéndez, Margarita; Laurents, Douglas V

    2012-03-01

    Gemini surfactants have two polar head groups and two hydrocarbon tails. Compared with conventional surfactants, geminis have much lower (μM vs. mM) critical micelle concentrations and possess slower (ms vs. μs) monomer <-- / --> micelle kinetics. The structure of the gemini surfactants studied is [HOCH(2)CH(2)-, CH(3)-, CH(3)(CH(2))(15)-N(+)-(CH(2))(s)-N(+)-(CH(2))(15)CH(3),-CH(3),-CH(2)CH(2)OH]·2Br(-) where s=4, 5, or 6. Our objective is to reveal the effect of these cationic gemini surfactants on the structure and stability of two model proteins: Ribonuclease A (RNase A) and Hen Egg White Lysozyme (HEWL). 2D (1)H NMR and Circular Dichroism (CD) spectroscopies show that the conformation of RNase A and HEWL is unaffected at low to neutral pH where these proteins are positively charged, although hydrogen exchange shows that RNase A's conformational stability is slightly lowered. At alkaline pH, where these proteins lose their net positive charge, fluorescence and CD spectroscopies and ITC experiments show that they do interact with gemini surfactants, and multiple protein•gemini complexes are observed. Based on the results, we conclude that these cationic gemini surfactants neither interact strongly with nor severely destabilize these well folded proteins in physiological conditions, and we advance that they can serve as useful membrane mimetics for studying the interactions between membrane components and positively charged proteins.

  14. The structure of salt bridges between Arg(+) and Glu(-) in peptides investigated with 2D-IR spectroscopy: Evidence for two distinct hydrogen-bond geometries.

    PubMed

    Huerta-Viga, Adriana; Amirjalayer, Saeed; Domingos, Sérgio R; Meuzelaar, Heleen; Rupenyan, Alisa; Woutersen, Sander

    2015-06-07

    Salt bridges play an important role in protein folding and in supramolecular chemistry, but they are difficult to detect and characterize in solution. Here, we investigate salt bridges between glutamate (Glu(-)) and arginine (Arg(+)) using two-dimensional infrared (2D-IR) spectroscopy. The 2D-IR spectrum of a salt-bridged dimer shows cross peaks between the vibrational modes of Glu(-) and Arg(+), which provide a sensitive structural probe of Glu(-)⋯Arg(+) salt bridges. We use this probe to investigate a β-turn locked by a salt bridge, an α-helical peptide whose structure is stabilized by salt bridges, and a coiled coil that is stabilized by intra- and intermolecular salt bridges. We detect a bidentate salt bridge in the β-turn, a monodentate one in the α-helical peptide, and both salt-bridge geometries in the coiled coil. To our knowledge, this is the first time 2D-IR has been used to probe tertiary side chain interactions in peptides, and our results show that 2D-IR spectroscopy is a powerful method for investigating salt bridges in solution.

  15. The structure of salt bridges between Arg+ and Glu- in peptides investigated with 2D-IR spectroscopy: Evidence for two distinct hydrogen-bond geometries

    NASA Astrophysics Data System (ADS)

    Huerta-Viga, Adriana; Amirjalayer, Saeed; Domingos, Sérgio R.; Meuzelaar, Heleen; Rupenyan, Alisa; Woutersen, Sander

    2015-06-01

    Salt bridges play an important role in protein folding and in supramolecular chemistry, but they are difficult to detect and characterize in solution. Here, we investigate salt bridges between glutamate (Glu-) and arginine (Arg+) using two-dimensional infrared (2D-IR) spectroscopy. The 2D-IR spectrum of a salt-bridged dimer shows cross peaks between the vibrational modes of Glu- and Arg+, which provide a sensitive structural probe of Glu-⋯Arg+ salt bridges. We use this probe to investigate a β-turn locked by a salt bridge, an α-helical peptide whose structure is stabilized by salt bridges, and a coiled coil that is stabilized by intra- and intermolecular salt bridges. We detect a bidentate salt bridge in the β-turn, a monodentate one in the α-helical peptide, and both salt-bridge geometries in the coiled coil. To our knowledge, this is the first time 2D-IR has been used to probe tertiary side chain interactions in peptides, and our results show that 2D-IR spectroscopy is a powerful method for investigating salt bridges in solution.

  16. 3D localized 2D ultrafast J-resolved magnetic resonance spectroscopy: in vitro study on a 7 T imaging system.

    PubMed

    Roussel, T; Giraudeau, P; Ratiney, H; Akoka, S; Cavassila, S

    2012-02-01

    2D Magnetic Resonance Spectroscopy (MRS) is a well known tool for the analysis of complicated and overlapped MR spectra and was therefore originally used for structural analysis. It also presents a potential for biomedical applications as shown by an increasing number of works related to localized in vivo experiments. However, 2D MRS suffers from long acquisition times due to the necessary collection of numerous increments in the indirect dimension (t(1)). This paper presents the first 3D localized 2D ultrafast J-resolved MRS sequence, developed on a small animal imaging system, allowing the acquisition of a 3D localized 2D J-resolved MRS spectrum in a single scan. Sequence parameters were optimized regarding Signal-to-Noise ratio and spectral resolution. Sensitivity and spatial localization properties were characterized and discussed. An automatic post-processing method allowing the reduction of artifacts inherent to ultrafast excitation is also presented. This sequence offers an efficient signal localization and shows a great potential for in vivo dynamic spectroscopy.

  17. Tubulin equilibrium unfolding followed by time-resolved fluorescence and fluorescence correlation spectroscopy

    PubMed Central

    Sánchez, Susana A.; Brunet, Juan E.; Jameson, David M.; Lagos, Rosalba; Monasterio, Octavio

    2004-01-01

    The pathway for the in vitro equilibrium unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been studied using several spectroscopic techniques, specifically circular dichroism (CD), two-photon Fluorescence Correlation Spectroscopy (FCS), and time-resolved fluorescence, including lifetime and dynamic polarization. The results show that tubulin unfolding is characterized by distinct processes that occur in different GdmCl concentration ranges. From 0 to 0.5 M GdmCl, a slight alteration of the tubulin heterodimer occurs, as evidenced by a small, but reproducible increase in the rotational correlation time of the protein and a sharp decrease in the secondary structure monitored by CD. In the range 0.5–1.5 M GdmCl, significant decreases in the steady-state anisotropy and average lifetime of the intrinsic tryptophan fluorescence occur, as well as a decrease in the rotational correlation time, from 48 to 26 nsec. In the same GdmCl range, the number of protein molecules (labeled with Alexa 488), as determined by two-photon FCS measurements, increases by a factor of two, indicating dissociation of the tubulin dimer into monomers. From 1.5 to 4 M GdmCl, these monomers unfold, as evidenced by the continual decrease in the tryptophan steady-state anisotropy, average lifetime, and rotational correlation time, concomitant with secondary structural changes. These results help to elucidate the unfolding pathway of the tubulin heterodimer and demonstrate the value of FCS measurements in studies on oligomeric protein systems. PMID:14691224

  18. Crystal structures and fluorescence properties of two 2D MnII/CdII trimellitic complexes containing terpyridine

    NASA Astrophysics Data System (ADS)

    Ren, Yixia; Chai, Hongmei; Hou, Xiangyang; Wang, Jijiang; Fu, Feng

    2015-12-01

    Hydrothermal reactions of manganese (II)/cadmium(II) salts with 1,2,4-trimellitic acid (H3tma) and 2,2‧:6‧,2-terpyridine (tpy) result in two novel complexes formulated with [M(Htma)(tpy)]·H2O (M = Mn(1) and Cd(2)). X-ray diffraction structural analyses of two complexes reveal they are isomorphic except for the different center metal ions and crystallize in the monoclinic crystal system of P(2)/n space group. The metal ion lies in a six-coordinated distorted octahedral environment coordinated with three Htma2- anions and one tpy ligand. There is an infinite two-dimensional rhombic network based on the metallic dimmers and Htma2- anions with the tpy ligands in void. Furthermore, the tpy ligands from the adjacent network weakly interact each other by π⋯π packing interactions into 3D supramolecular structure. The fluorescence properties could be assigned to the π - π* transition of organic ligands.

  19. Expression of green fluorescent protein in human foreskin fibroblasts for use in 2D and 3D culture models.

    PubMed

    Chao, Jie; Peña, Tiffany; Heimann, Dean G; Hansen, Chris; Doyle, David A; Yanala, Ujwal R; Guenther, Timothy M; Carlson, Mark A

    2014-01-01

    The availability of fibroblasts that express green fluorescent protein (GFP) would be of interest for the monitoring of cell growth, migration, contraction, and other processes within the fibroblast-populated collagen matrix and other culture systems. A plasmid lentiviral vector-GFP (pLV-GFP) was utilized for gene delivery to produce primary human foreskin fibroblasts (HFFs) that stably express GFP. Cell morphology, cell migration, and collagen contraction were compared between nontransduced HFFs and transduced GFP-HFFs; no differences were observed. Immunocytochemical staining showed no differences in cell morphology between nontransduced and GFP-HFFs in both two-dimensional and three-dimensional culture systems. Furthermore, there was no significant difference in cellular population growth within the collagen matrix populated with nontransduced vs. GFP-HFFs. Within the limits of our assays, we conclude that transduction of GFP into HFFs did not alter the observed properties of HFFs compared with nontransduced fibroblasts. The GFP-HFFs may represent a new tool for the convenient monitoring of living primary fibroblast processes in two-dimensional or three-dimensional culture.

  20. Fluorescence quantum yield measurement in nanoparticle-fluorophore systems by thermal lens spectroscopy

    NASA Astrophysics Data System (ADS)

    Ferreira, M.; Piscitelli, V.

    2016-04-01

    Metallic nanoparticles have been used as a way to tailor the fluorescence properties like quantum yield, but regular fluorescence quantum yield measurements have to counter the reflection and dispersion of a sample for an accurate result. Thermal lens spectroscopy is a good alternative to resolve this problem because doesn't measure the fluorescence intensity but the heat generated by absorption. We studied the changes induced by silver nanoparticles, generated by laser ablation, in the fluorescence peak and quantum yield of Rhodamine B. We fund that the silver nanoparticles lowered the fluorescence peak and quenched the fluorescence of the Rhodamine B and how much is quenched also depends on its concentration.

  1. Rovibrational analysis of the ethylene isotopologue 13C2D4 by high-resolution Fourier transform infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Tan, T. L.; Gabona, M. G.; Godfrey, Peter D.; McNaughton, Don

    2015-01-01

    The Fourier transform infrared (FTIR) spectrum of the unperturbed a-type ν12 band of 13C2D4 was recorded at an unapodized resolution of 0.0063 cm-1 between 1000 and 1140 cm-1 for a rovibrational analysis. By assigning and fitting a total of 2068 infrared transitions using a Watson's A-reduced and S-reduced Hamiltonians in the Ir representation, rovibrational constants for the upper state (ν12 = 1) up to five quartic centrifugal distortion terms were derived for the first time. The root-mean-square (rms) deviation of the fits was 0.00034 cm-1 both in the A-reduction and S-reduction Hamiltonian. The ground state rovibrational constants of 13C2D4 in the A-reduced and S-reduced Hamiltonians were also determined for the first time by a fit of 985 combination-differences from the present infrared measurements, with rms deviation of 0.00036 cm-1. The ν12 band centre of 13C2D4 was at 1069.970824(17) cm-1 and at 1069.970799(17) cm-1 for the A-reduced and S-reduced Hamiltonians respectively. The ground state constants of 13C2D4 from this experimental work are in close agreement to those derived from theoretical calculations using the B3LYP/cc-pVTZ, MP2/cc-pVTZ, and CSSD(T)/cc-pVTZ levels of theory.

  2. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    DOE PAGES

    Laurence, Ted A.; Ly, Sonny; Bourguet, Feliza; ...

    2014-08-14

    Fluorescence correlation spectroscopy (FCS) is an important technique for studying biochemical interactions dynamically that may be used in vitro and in cell-based studies. It is generally claimed that FCS may only be used at nM concentrations. We show that this general consensus is incorrect and that the limitation to nM concentrations is not fundamental but due to detector limits as well as laser fluctuations. With a high count rate detector system and applying laser fluctuation corrections, we demonstrate FCS measurements up to 38 μM with the same signal-to-noise as at lower concentrations. Optical nanoconfinement approaches previously used to increase themore » concentration range of FCS are not necessary, and further increases above 38 μM may be expected using detectors and detector arrays with higher saturation rates and better laser fluctuation corrections. This approach greatly widens the possibilities of dynamic measurements of biochemical interactions using FCS at physiological concentrations.« less

  3. Inference of protein diffusion probed via fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Tsekouras, Konstantinos

    2015-03-01

    Fluctuations are an inherent part of single molecule or few particle biophysical data sets. Traditionally, ``noise'' fluctuations have been viewed as a nuisance, to be eliminated or minimized. Here we look on how statistical inference methods - that take explicit advantage of fluctuations - have allowed us to draw an unexpected picture of single molecule diffusional dynamics. Our focus is on the diffusion of proteins probed using fluorescence correlation spectroscopy (FCS). First, we discuss how - in collaboration with the Bustamante and Marqusee labs at UC Berkeley - we determined using FCS data that individual enzymes are perturbed by self-generated catalytic heat (Riedel et al, Nature, 2014). Using the tools of inference, we found how distributions of enzyme diffusion coefficients shift in the presence of substrate revealing that enzymes performing highly exothermic reactions dissipate heat by transiently accelerating their center of mass following a catalytic reaction. Next, when molecules diffuse in the cell nucleus they often appear to diffuse anomalously. We analyze FCS data - in collaboration with Rich Day at the IU Med School - to propose a simple model for transcription factor binding-unbinding in the nucleus to show that it may give rise to apparent anomalous diffusion. Here inference methods extract entire binding affinity distributions for the diffusing transcription factors, allowing us to precisely characterize their interactions with different components of the nuclear environment. From this analysis, we draw key mechanistic insight that goes beyond what is possible by simply fitting data to ``anomalous diffusion'' models.

  4. Subdiffusive molecular motion in nanochannels observed by fluorescence correlation spectroscopy.

    PubMed

    De Santo, Ilaria; Causa, Filippo; Netti, Paolo A

    2010-02-01

    The influence of confinement on biomolecule motion in glass channels of nanometric height has been investigated with fluorescence correlation spectroscopy (FCS). We measured intrachannel molecule diffusion time and concentration based on a single-component diffusion model as a function of molecule size to channel height (r(g)/h). Poly(ethylene glycol) (PEG) of 20 kDa and dextran of 40 kDa showed a reduction of their diffusion coefficients of almost 1 order of magnitude when nanochannel height approached probe diameter, whereas rhodamine 6G (Rh6G) was shown to be almost unaffected from confinement. Subdiffusive motion has been proven for flexible molecules in nanochannels, and deviations toward a square root dependence of mobility with time for confinement up to molecule size r(g)/h approximately 0.5 were registered. Diffusion coefficient time dependence has been evaluated and described with a model that accounts for diffusion time increase due to molecule rearrangements related to molecule flexibility and surface interactions dynamics. The evaluation of the subdiffusive mode and the key parameters extracted at the single-molecule level of partitioning, intrachannel diffusion time, desorption time, and binding probability at surfaces can be exploited for the engineering of bioanalytic nanodevices.

  5. Fluorescence Correlation Spectroscopy and Nonlinear Stochastic Reaction-Diffusion

    SciTech Connect

    Del Razo, Mauricio; Pan, Wenxiao; Qian, Hong; Lin, Guang

    2014-05-30

    The currently existing theory of fluorescence correlation spectroscopy (FCS) is based on the linear fluctuation theory originally developed by Einstein, Onsager, Lax, and others as a phenomenological approach to equilibrium fluctuations in bulk solutions. For mesoscopic reaction-diffusion systems with nonlinear chemical reactions among a small number of molecules, a situation often encountered in single-cell biochemistry, it is expected that FCS time correlation functions of a reaction-diffusion system can deviate from the classic results of Elson and Magde [Biopolymers (1974) 13:1-27]. We first discuss this nonlinear effect for reaction systems without diffusion. For nonlinear stochastic reaction-diffusion systems there are no closed solutions; therefore, stochastic Monte-Carlo simulations are carried out. We show that the deviation is small for a simple bimolecular reaction; the most significant deviations occur when the number of molecules is small and of the same order. Extending Delbrück-Gillespie’s theory for stochastic nonlinear reactions with rapidly stirring to reaction-diffusion systems provides a mesoscopic model for chemical and biochemical reactions at nanometric and mesoscopic level such as a single biological cell.

  6. Fluorescence Correlation Spectroscopy Evidence for Structural Heterogeneity in Ionic Liquids

    SciTech Connect

    Guo, Jianchang; Baker, Gary A; Hillesheim, Patrick C; Dai, Sheng; Shaw, Robert W; Mahurin, Shannon Mark

    2011-01-01

    Self-aggregation in room temperature ionic liquids (RTILs) has been a subject of intense interest in recent years. In this work, we provide new experimental evidence for chain length-dependent self-aggregation in RTILs using fluorescence correlation spectroscopy (FCS). In studying a homologous series of N-alkyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl) imide, [CnMPy][Tf2N] RTILs of varying alkyl chain length (n = 3, 4, 6, 8, and 10), biphasic rhodamine 6G solute diffusion dynamics were observed; both the fast and slow diffusion coefficients decrease with increasing alkyl chain length, with the relative contribution from slower diffusion increasing for longer-chained [CnMPy][Tf2N]. We propose that the biphasic diffusion dynamics originate from self-aggregation of the nonpolar alkyl chains in the cationic [CnMPy]+. The presence of this local liquid structuring provides important insight into the behavior of RTILs relevant to their application in photovoltaics, fuel cells, and batteries.

  7. The Intermediate Scattering Function in Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Guerra, Rodrigo; Andrews, Ballard; Sen, Pabitra

    2006-03-01

    We formulate the autocorrelation function for Fluorescence Correlation Spectroscopy (FCS) GD(τ) in reciprocal space in terms of the of the Intermediate Scattering Function ISF(k,t) and the fourier transform of the Optical Response Function ORF(k). In this way we may extend the use of FCS to processes that have been studied using NMR, DLS, and neutron scattering. This formalism is useful for the complicated propagators involved in confined systems and in the study of diffusion in cells: where diffusion is either restricted or permeation through membrane is important. Calculations in k-space produce approximate expressions for the ORF using cumulant expansions that are accurate for small wavevectors. This provides descriptions for longer timescales better suited for studying time-dependent diffusion ISF(k,t)->exp[-tD(t)k^2] and provides a natural separation of contributions from system dynamics and from optical artifacts and aberrations. We will show an explicit derivation of a semi-analytical fit function for free diffusion based on standard electromagnetic analysis of a confocal optical apparatus. This fit function is then used to analyze a representative data set and has no free fit parameters other than the diffusion constant.

  8. Structured illumination fluorescence correlation spectroscopy for velocimetry in Zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Pozzi, Paolo; Rossetti, Leone; Sironi, Laura; Freddi, Stefano; D'Alfonso, Laura; Caccia, Michele; Bouzin, Margaux; Collini, Maddalena; Chirico, Giuseppe

    2013-02-01

    The vascular system of Zebrafish embryos is studied by means of Fluorescence Correlation and Image Correlation Spectroscopy. The long term project addresses biologically relevant issues concerning vasculogenesis and cardiogenesis and in particular mechanical interaction between blood flow and endothelial cells. To this purpose we use Zebrafish as a model system since the transparency of its embryos facilitates morphological observation of internal organs in-vivo. The correlation analysis provides quantitative characterization of fluxes in blood vessels in vivo. We have pursued and compared two complementary routes. In a first one we developed a two-spots two-photon setup in which the spots are spaced at adjustable micron-size distances (1-40 μm) along a vessel and the endogenous (autofluorescence) or exogenous (dsRed transgenic erythrocytes) signal is captured with an EM-CCD and cross-correlated. In this way we are able to follow the morphology of the Zebrafish embryo, simultaneously measure the heart pulsation, the velocity of red cells and of small plasma proteins. These data are compared to those obtained by image correlations on Zebrafish vessels. The two methods allows to characterize the motion of plasma fluids and erythrocytes in healthy Zebrafish embryos to be compared in the future to pathogenic ones.

  9. Vibrational dynamics and solvatochromism of the label SCN in various solvents and hemoglobin by time dependent IR and 2D-IR spectroscopy.

    PubMed

    van Wilderen, Luuk J G W; Kern-Michler, Daniela; Müller-Werkmeister, Henrike M; Bredenbeck, Jens

    2014-09-28

    We investigated the characteristics of the thiocyanate (SCN) functional group as a probe of local structural dynamics for 2D-IR spectroscopy of proteins, exploiting the dependence of vibrational frequency on the environment of the label. Steady-state and time-resolved infrared spectroscopy are performed on the model compound methylthiocyanate (MeSCN) in solvents of different polarity, and compared to data obtained on SCN as a local probe introduced as cyanylated cysteine in the protein bovine hemoglobin. The vibrational lifetime of the protein label is determined to be 37 ps, and its anharmonicity is observed to be lower than that of the model compound (which itself exhibits solvent-independent anharmonicity). The vibrational lifetime of MeSCN generally correlates with the solvent polarity, i.e. longer lifetimes in less polar solvents, with the longest lifetime being 158 ps. However, the capacity of the solvent to form hydrogen bonds complicates this simplified picture. The long lifetime of the SCN vibration is in contrast to commonly used azide labels or isotopically-labeled amide I and better suited to monitor structural rearrangements by 2D-IR spectroscopy. We present time-dependent 2D-IR data on the labeled protein which reveal an initially inhomogeneous structure around the CN oscillator. The distribution becomes homogeneous after 5 picoseconds so that spectral diffusion has effectively erased the 'memory' of the CN stretching frequency. Therefore, the 2D-IR data of the label incorporated in hemoglobin demonstrate how SCN can be utilized to sense rearrangements in the local structure on a picosecond timescale.

  10. Fluorescence fluctuation spectroscopy: ushering in a new age of enlightenment for cellular dynamics

    PubMed Central

    Jameson, David M.; Ross, Justin A.; Albanesi, Joseph P.

    2011-01-01

    Originally developed for applications in physics and physical chemistry, fluorescence fluctuation spectroscopy is becoming widely used in cell biology. This review traces the development of the method and describes some of the more important applications. Specifically, the methods discussed include fluorescence correlation spectroscopy (FCS), scanning FCS, dual color cross-correlation FCS, the photon counting histogram and fluorescence intensity distribution analysis approaches, the raster scanning image correlation spectroscopy method, and the Number and Brightness technique. The physical principles underlying these approaches will be delineated, and each of the methods will be illustrated using examples from the literature. PMID:21547245

  11. Fluorescence fluctuation spectroscopy: ushering in a new age of enlightenment for cellular dynamics.

    PubMed

    Jameson, David M; Ross, Justin A; Albanesi, Joseph P

    2009-09-01

    Originally developed for applications in physics and physical chemistry, fluorescence fluctuation spectroscopy is becoming widely used in cell biology. This review traces the development of the method and describes some of the more important applications. Specifically, the methods discussed include fluorescence correlation spectroscopy (FCS), scanning FCS, dual color cross-correlation FCS, the photon counting histogram and fluorescence intensity distribution analysis approaches, the raster scanning image correlation spectroscopy method, and the Number and Brightness technique. The physical principles underlying these approaches will be delineated, and each of the methods will be illustrated using examples from the literature.

  12. Excimer laser fragmentation fluorescence spectroscopy for real-time monitoring of combustion generated pollutants

    NASA Astrophysics Data System (ADS)

    Damm, Christopher John

    Toxic pollutant emissions from combustion pose a hazard to public and environmental health. Better diagnostic techniques would benefit emissions monitoring programs and aid research aimed at understanding toxic pollutant formation and behavior. Excimer Laser Fragmentation Fluorescence Spectroscopy (ELFFS) provides sensitive, real-time, in situ measurements of several important combustion related pollutants. This thesis demonstrates the capabilities of ELFFS for detecting amines in combustion exhausts and carbonaceous particulate matter from engines. ELFFS photofragments target species using a 193 nm excimer laser to form fluorescent signature species. The NH (A--X) band at 336 nm is used to monitor ammonia, ammonium nitrate and ammonium sulfate. There are no major interferences in this spectral region. The sensitivity is approximately 100 ppb (1 second measurement) for ammonia in post flame gases and 100 ppb (mole fraction) for ammonium nitrate/sulfate in ambient air. Quenching of NH by the major combustion products does not limit the applicability of the detection method. Fluorescence from excited carbon atoms at 248 nm (1P 0 → 1S0) following photofragmentation measures particulate matter in a two-stroke gasoline engine and a four-stroke diesel engine. Fluorescence from CH (A2Delta → X 2pi, 431 nm) C2 (d3pig → a3piu, 468 nm) fragments is also observed. The atomic carbon fluorescence signal is proportional to the mass concentration of particles in the laser interrogation region. The 100-shot (1 second) detection limit for particles in the two-stroke gasoline engine exhaust is 0.5 ppb (volume fraction). The 100-shot detection limit for four-stroke diesel particulate matter is 0.2 ppb. Interferences from carbon monoxide and carbon dioxide are negligible. The ratios of atomic carbon, C2, and CH peaks provide information on the molecular forms of compounds condensed on or contained within the particles measured. The C/C2 signal ratio can be used to distinguish

  13. 2D IR spectroscopy at 100 kHz utilizing a Mid-IR OPCPA laser source.

    PubMed

    Luther, Bradley M; Tracy, Kathryn M; Gerrity, Michael; Brown, Susannah; Krummel, Amber T

    2016-02-22

    We present a 100 kHz 2D IR spectrometer. The system utilizes a ytterbium all normal dispersion fiber oscillator as a common source for the pump and seed beams of a MgO:PPLN OPCPA. The 1030 nm OPCPA pump is generated by amplification of the oscillator in cryocooled Yb:YAG amplifiers, while the 1.68 μm seed is generated in a OPO pumped by the oscillator. The OPCPA outputs are used in a ZGP DFG stage to generate 4.65 μm pulses. A mid-IR pulse shaper delivers pulse pairs to a 2D IR spectrometer allowing for data collection at 100 kHz.

  14. Terahertz Spectroscopy of the Bending Vibrations of Acetylene 12C2H2 and 12C2D2

    NASA Astrophysics Data System (ADS)

    Yu, Shanshan; Drouin, B.; Pearson, J.

    2009-12-01

    Several fundamental interstellar molecules, e.g., C2H2, CH4 and C3, are completely symmetric molecules and feature no permanent dipole moment and no pure rotation spectrum. As a result they have only previously been observed in the infrared. However, directly observing them with the rest of the molecular column especially when the source is spatially resolved would be very valuable in understanding chemical evolution. Vibrational difference bands provide a means to detect symmetric molecules with microwave precision using terahertz techniques. Herschel, SOFIA and ALMA have the potential to identify a number of vibrational difference bands of light symmetric species. This paper reports laboratory results on 12C2H2 and 12C2D2. Symmetric acetylene isotopologues have two bending modes, the trans bending and the cis bending. Their difference bands are allowed and occur in the microwave, terahertz, and far-infrared wavelengths, with band origins at 3500 GHz for 12C2H2 and 900 GHz for 12C2D2. Twenty 12C2H2 P branch high-J transitions and two hundred and fifty-one 12C2D2 P Q and R branch transitions have been measured in the 0.2 - 1.6 THz region with precision of 50 to 100 kHz. These lines were modeled together with prior data on the pure bending levels. Significantly improved molecular parameters were obtained for 12C2H2 and 12C2D2 with the combined data set, and new frequency and intensity predictions were made to support astrophysics applications. The research was performed at the Jet Propulsion Laboratory, California Institute of Technology, under contract with the National Aeronautics and Space Administration. S. Y. was supported by an appointment to the NASA Postdoctoral Program, administrated by Oak Ridge Associated Universities through a contract with NASA.

  15. Time-resolved fluorescence spectroscopy of oil spill detected by ocean lidar

    NASA Astrophysics Data System (ADS)

    Li, Xiao-long; Chen, Yong-hua; Li, Jie; Jiang, Jingbo; Ni, Zuotao; Liu, Zhi-shen

    2016-10-01

    Based on time-resolved fluorescence of oils, an oceanographic fluorescence Lidar was designed to identify oil pollutions. A third harmonic (at 355nm) of Nd:YAG laser is used as the excitation source, and the fluorescence intensities and lifetimes of oil fluorescence at wavelength from 380 nm to 580 nm are measured by an intensified CCD (ICCD). In the experiments, time-resolved fluorescence spectra of 20 oil samples, including crude oils, fuel oils, lubricating oil, diesel oils and gasoline, are analyzed to discuss fluorescence spectral characteristics of samples for oil classification. The spectral characteristics of oil fluorescence obtained by ICCD with delay time of 2 ns, 4 ns, and 6 ns were studied by using the principal component analysis (PCA) method. Moreover, an efficient method is used to improve the recognition rate of the oil spill types, through enlarging spectral differences of oil fluorescence at different delay times. Experimental analysis shows that the optimization method can discriminate between crude oil and fuel oil, and a more accurate classification of oils is obtained by time-resolved fluorescence spectroscopy. As the result, comparing to traditional fluorescence spectroscopy, a higher recognition rate of oil spill types is achieved by time-resolved fluorescence spectroscopy which is also a feasibility technology for Ocean Lidar.

  16. 2D-IR spectroscopy of the sulfhydryl band of cysteines in the hydrophobic core of proteins.

    PubMed

    Koziński, M; Garrett-Roe, S; Hamm, P

    2008-06-26

    We investigate the sulfhydryl band of cysteines as a new chromophore for two-dimensional IR (2D-IR) studies of the structure and dynamics of proteins. Cysteines can be put at almost any position in a protein by standard methods of site-directed mutagenesis and, hence, have the potential to be an extremely versatile local probe. Although being a very weak absorber in aqueous environment, the sulfhydryl group gets strongly polarized when situated in an alpha-helix inside the hydrophobic core of a protein because of a strong hydrogen bond to the backbone carbonyl group. The extinction coefficient (epsilon=150 M(-1) cm(-1)) then is sufficiently high to perform detailed 2D-IR studies even at low millimolar concentrations. Using porcine (carbonmonoxy)hemoglobin as an example, which contains two such cysteines in its wild-type form, we demonstrate that spectral diffusion deduced from the 2D-IR line shapes reports on the overall-breathing of the corresponding alpha-helix. The vibrational lifetime of the sulfhydryl group (T1 approximately 6 ps) is considerably longer than that of the much more commonly used amide I mode (approximately 1.0 ps), thereby significantly extending the time window in which spectral diffusion processes can be observed. The experiments are accompanied by molecular dynamics simulations revealing a good overall agreement.

  17. Two dimensional laser induced fluorescence spectroscopy: A powerful technique for elucidating rovibronic structure in electronic transitions of polyatomic molecules

    NASA Astrophysics Data System (ADS)

    Gascooke, Jason R.; Alexander, Ula N.; Lawrance, Warren D.

    2011-05-01

    We demonstrate the power of high resolution, two dimensional laser induced fluorescence (2D-LIF) spectroscopy for observing rovibronic transitions of polyatomic molecules. The technique involves scanning a tunable laser over absorption features in the electronic spectrum while monitoring a segment, in our case 100 cm-1 wide, of the dispersed fluorescence spectrum. 2D-LIF images separate features that overlap in the usual laser induced fluorescence spectrum. The technique is illustrated by application to the S1-S0 transition in fluorobenzene. Images of room temperature samples show that overlap of rotational contours by sequence band structure is minimized with 2D-LIF allowing a much larger range of rotational transitions to be observed and high precision rotational constants to be extracted. A significant advantage of 2D-LIF imaging is that the rotational contours separate into their constituent branches and these can be targeted to determine the three rotational constants individually. The rotational constants determined are an order of magnitude more precise than those extracted from the analysis of the rotational contour and we find the previously determined values to be in error by as much as 5% [G. H. Kirby, Mol. Phys. 19, 289 (1970), 10.1080/00268977000101291]. Comparison with earlier ab initio calculations of the S0 and S1 geometries [I. Pugliesi, N. M. Tonge, and M. C. R. Cockett, J. Chem. Phys. 129, 104303 (2008), 10.1063/1.2970092] reveals that the CCSD/6-311G** and RI-CC2/def2-TZVPP levels of theory predict the rotational constants, and hence geometries, with comparable accuracy. Two ground state Fermi resonances were identified by the distinctive patterns that such resonances produce in the images. 2D-LIF imaging is demonstrated to be a sensitive method capable of detecting weak spectral features, particularly those that are otherwise hidden beneath stronger bands. The sensitivity is demonstrated by observation of the three isotopomers of fluorobenzene

  18. Two dimensional laser induced fluorescence spectroscopy: a powerful technique for elucidating rovibronic structure in electronic transitions of polyatomic molecules.

    PubMed

    Gascooke, Jason R; Alexander, Ula N; Lawrance, Warren D

    2011-05-14

    We demonstrate the power of high resolution, two dimensional laser induced fluorescence (2D-LIF) spectroscopy for observing rovibronic transitions of polyatomic molecules. The technique involves scanning a tunable laser over absorption features in the electronic spectrum while monitoring a segment, in our case 100 cm(-1) wide, of the dispersed fluorescence spectrum. 2D-LIF images separate features that overlap in the usual laser induced fluorescence spectrum. The technique is illustrated by application to the S(1)-S(0) transition in fluorobenzene. Images of room temperature samples show that overlap of rotational contours by sequence band structure is minimized with 2D-LIF allowing a much larger range of rotational transitions to be observed and high precision rotational constants to be extracted. A significant advantage of 2D-LIF imaging is that the rotational contours separate into their constituent branches and these can be targeted to determine the three rotational constants individually. The rotational constants determined are an order of magnitude more precise than those extracted from the analysis of the rotational contour and we find the previously determined values to be in error by as much as 5% [G. H. Kirby, Mol. Phys. 19, 289 (1970)]. Comparison with earlier ab initio calculations of the S(0) and S(1) geometries [I. Pugliesi, N. M. Tonge, and M. C. R. Cockett, J. Chem. Phys. 129, 104303 (2008)] reveals that the CCSD∕6-311G∗∗ and RI-CC2∕def2-TZVPP levels of theory predict the rotational constants, and hence geometries, with comparable accuracy. Two ground state Fermi resonances were identified by the distinctive patterns that such resonances produce in the images. 2D-LIF imaging is demonstrated to be a sensitive method capable of detecting weak spectral features, particularly those that are otherwise hidden beneath stronger bands. The sensitivity is demonstrated by observation of the three isotopomers of fluorobenzene-d(1) in natural abundance in

  19. Four divalent transition metal carboxyarylphosphonate compounds: Hydrothermal synthesis, structural chemistry and generalized 2D FTIR correlation spectroscopy studies

    SciTech Connect

    Lei Ran; Chai Xiaochuan; Mei Hongxin; Zhang Hanhui; Chen Yiping; Sun Yanqiong

    2010-07-15

    Four divalent transition metal carboxyarylphosphonates, [Ni(4,4'-bipy)H{sub 2}L{sup 1}(HL{sup 1}){sub 2}(H{sub 2}O){sub 2}].2H{sub 2}O 1, [Ni{sub 2}(4,4'-bipy)(L{sup 2})(OH)(H{sub 2}O){sub 2}].3H{sub 2}O 2, Mn(phen){sub 2}(H{sub 2}L{sup 1}){sub 2}3 and Mn(phen)(HL{sup 2}) 4 (H{sub 3}L{sup 1}=p-H{sub 2}O{sub 3}PCH{sub 2}-C{sub 6}H{sub 4}-COOH, H{sub 3}L{sup 2}=m-H{sub 2}O{sub 3}PCH{sub 2}-C{sub 6}H{sub 4}-COOH, 4,4'-bipy=4,4'-bipyridine, phen=1,10-phenanthroline) were synthesized under hydrothermal conditions. 1 features 1D linear chains built from Ni(II) ions bridging 4,4'-bipy. In 2, neighboring Ni{sub 4} cluster units are connected by pairs of H{sub 3}L{sup 2} ligands to form 1D double-crankshaft chains, which are interconnected by pairs of 4,4'-bipy into 2D sheets. 3 exhibits 2D supramolecular layers via the R{sub 2}{sup 2}(8) ringed hydrogen bonding units. 4 has 1D ladderlike chains, in which the 4-membered rings are cross-linked by the organic moieties of the H{sub 3}L{sup 2} ligands. Additionally, 2D FTIR correlation analysis is applied with thermal and magnetic perturbation to clarify the structural changes of functional groups from H{sub 3}L{sup 1} and H{sub 3}L{sup 2} ligands in the compounds more efficiently. - Graphical abstract: A series of divalent transition metal carboxyarylphosphonate compounds were synthesized under hydrothermal conditions. The figure displays 2D sheet structure with large windows in compound 2.

  20. Unraveling the dynamics and structure of functionalized self-assembled monolayers on gold using 2D IR spectroscopy and MD simulations

    PubMed Central

    Yan, Chang; Yuan, Rongfeng; Pfalzgraff, William C.; Nishida, Jun; Wang, Lu; Markland, Thomas E.; Fayer, Michael D.

    2016-01-01

    Functionalized self-assembled monolayers (SAMs) are the focus of ongoing investigations because they can be chemically tuned to control their structure and dynamics for a wide variety of applications, including electrochemistry, catalysis, and as models of biological interfaces. Here we combine reflection 2D infrared vibrational echo spectroscopy (R-2D IR) and molecular dynamics simulations to determine the relationship between the structures of functionalized alkanethiol SAMs on gold surfaces and their underlying molecular motions on timescales of tens to hundreds of picoseconds. We find that at higher head group density, the monolayers have more disorder in the alkyl chain packing and faster dynamics. The dynamics of alkanethiol SAMs on gold are much slower than the dynamics of alkylsiloxane SAMs on silica. Using the simulations, we assess how the different molecular motions of the alkyl chain monolayers give rise to the dynamics observed in the experiments. PMID:27044113

  1. Protein oligomerization monitored by fluorescence fluctuation spectroscopy: Self-assembly of Rubisco activase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A methodology is presented to characterize complex protein assembly pathways by fluorescence correlation spectroscopy. We have derived the total autocorrelation function describing the behavior of mixtures of labeled and unlabeled protein under equilibrium conditions. Our modeling approach allows us...

  2. Two-dimensional correlation spectroscopy (2D-COS) variable selection for near-infrared microscopy discrimination of meat and bone meal in compound feed.

    PubMed

    Lü, Chengxu; Chen, Longjian; Yang, Zengling; Liu, Xian; Han, Lujia

    2014-01-01

    This article presents a novel method for combining auto-peak and cross-peak information for sensitive variable selection in synchronous two-dimensional correlation spectroscopy (2D-COS). This variable selection method is then applied to the case of near-infrared (NIR) microscopy discrimination of meat and bone meal (MBM). This is of important practical value because MBM is currently banned in ruminate animal compound feed. For the 2D-COS analysis, a set of NIR spectroscopy data of compound feed samples (adulterated with varying concentrations of MBM) was pretreated using standard normal variate and detrending (SNVD) and then mapped to the 2D-COS synchronous matrix. For the auto-peak analysis, 12 main sensitive variables were identified at 6852, 6388, 6320, 5788, 5600, 5244, 4900, 4768, 4572, 4336, 4256, and 4192 cm(-1). All these variables were assigned their specific spectral structure and chemical component. For the cross-peak analysis, these variables were divided into two groups, each group containing the six sensitive variables. This grouping resulted in a correlation between the spectral variables that was in accordance with the chemical-component content of the MBM and compound feed. These sensitive variables were then used to build a NIR microscopy discrimination model, which yielded a 97% correct classification. Moreover, this method detected the presence of MBM when its concentration was less than 1% in an adulterated compound feed sample. The concentration-dependent 2D-COS-based variable selection method developed in this study has the unique advantages of (1) introducing an interpretive aspect into variable selection, (2) substantially reducing the complexity of the computations, (3) enabling the transferability of the results to discriminant analysis, and (4) enabling the efficient compression of spectral data.

  3. Multiple stimulated emission fluorescence photoacoustic sensing and spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Gaoming; Gao, Fei; Qiu, Yishen; Feng, Xiaohua; Zheng, Yuanjin

    2016-07-01

    Multiple stimulated emission fluorescence photoacoustic (MSEF-PA) phenomenon is demonstrated in this letter. Under simultaneous illumination of pumping light and stimulated emission light, the fluorescence emission process is speeded up by the stimulated emission effect. This leads to nonlinear enhancement of photoacoustic signal while the quantity of absorbed photons is more than that of fluorescent molecules illuminated by pumping light. The electronic states' specificity of fluorescent molecular can also be labelled by the MSEF-PA signals, which can potentially be used to obtain fluorescence excitation spectrum in deep scattering tissue with nonlinearly enhanced photoacoustic detection. In this preliminary study, the fluorescence excitation spectrum is reconstructed by MSEF-PA signals through sweeping the wavelength of exciting light, which confirms the theoretical derivation well.

  4. Exploring doxorubicin localization in eluting TiO2 nanotube arrays through fluorescence correlation spectroscopy analysis.

    PubMed

    De Santo, Ilaria; Sanguigno, Luigi; Causa, Filippo; Monetta, Tullio; Netti, Paolo A

    2012-11-07

    Drug elution properties of TiO(2) nanotube arrays have been largely investigated by means of solely macroscopic observations. Controversial elution performances have been reported so far and a clear comprehension of these phenomena is still missing as a consequence of a lack of molecular investigation methods. Here we propose a way to discern drug elution properties of nanotubes through the evaluation of drug localization by Fluorescence Correlation Spectroscopy (FCS) analysis. We verified this method upon doxorubicin elution from differently loaded TiO(2) nanotubes. Diverse elution profiles were obtained from nanotubes filled by soaking and wet vacuum impregnation methods. Impregnated nanotubes controlled drug diffusion up to thirty days, while soaked samples completed elution in seven days. FCS analysis of doxorubicin motion in loaded nanotubes clarified that more than 90% of drugs dwell preferentially in inter-nanotube spaces in soaked samples due to decorrelation in a 2D fashion, while a 97% fraction of molecules showed 1D mobility ascribable to displacements along the nanotube vertical axis of wet vacuum impregnated nanotubes. The diverse drug localizations inferred from FCS measurements, together with distinct drug-surface interaction strengths resulting from diverse drug filling techniques, could explain the variability in elution kinetics.

  5. Application of fluorescence spectroscopy and chemometrics in the evaluation of processed cheese during storage.

    PubMed

    Christensen, J; Povlsen, V T; Sørensen, J

    2003-04-01

    Front face fluorescence spectroscopy is applied for an evaluation of the stability of processed cheese during storage. Fluorescence landscapes with excitation from 240 to 360 nm and emission in the range of 275 to 475 nm were obtained from cheese samples stored in darkness and light in up to 259 d, at 5, 20 and 37 degrees C, respectively. Parallel factor (PARAFAC) analysis of the fluorescence landscapes exhibits four fluorophores present in the cheese, all related to the storage conditions. The chemometric analysis resolves the fluorescence signal into excitation and emission profiles of the pure fluorescent compounds, which are suggested to be tryptophan, vitamin A and a compound derived from oxidation. Thus, it is concluded that fluorescence spectroscopy in combination with chemometrics has a potential as a fast method for monitoring the stability of processed cheese.

  6. The Application Of Picosecond-Resolved Fluorescence Spectroscopy In The Study Of Flavins And Flavoproteins

    NASA Astrophysics Data System (ADS)

    Visser, Antonie J.; van Hoek, Arie

    1988-06-01

    Picosecond relaxation processes of flavins and flavoproteins were investigated with mode-locked and synchronously pumped lasers as source of excitation and time-correlated single photon counting in detection. Free flavin rotational correlation times of 80-150 ps (values depending on the flavin derivative used) could be precisely determined. Picosecond-resolved fluorescence of the flavin bound in the electron-carrier protein flavodoxin from Desulfovibrio vulgaris yields a fluorescence lifetime component of 30 ps in the fluorescence decay. Time-resolved tryptophan fluorescence in flavodoxin exhibits a short lifetime component, which is attributed in part to energy transfer from tryptophan to flavin. Three-dimensional fluorescence spectroscopy and fluorescence anisotropy decay analysis of the two tryptophan residues in flavodoxin provide new evidence for specific flavin-tryptophan interaction. Finally, picosecond-resolved spectroscopy enables the direct measurement of energy transfer between two different chromophores in a protein, from which topographical details can be inferred.

  7. Excitation-emission matrices and synchronous fluorescence spectroscopy for the diagnosis of gastrointestinal cancers

    NASA Astrophysics Data System (ADS)

    Genova, Ts; Borisova, E.; Penkov, N.; Vladimirov, B.; Zhelyazkova, A.; Avramov, L.

    2016-06-01

    We report the development of an improved fluorescence technique for cancer diagnostics in the gastrointestinal tract. We investigate the fluorescence of ex vivo colorectal (cancerous and healthy) tissue samples using excitation-emission matrix (EEM) and synchronous fluorescence spectroscopy (SFS) steady-state approaches. The obtained results are processed for revealing characteristic fluorescence spectral features with a valuable diagnostic meaning. The main tissue fluorophores, contributing to the observed fluorescence, are tyrosine, tryptophan, NADH, FAD, collagen and elastin. Based on the results of the Mann-Whitney test as useful parameters for differentiation of gastrointestinal cancer from normal mucosa, we suggest using excitation wavelengths in the range 300 - 360 nm for fluorescence spectroscopy and wavelengths intervals of 60 nm and 90 nm for SFS.

  8. Polarization shaping in the mid-IR and polarization-based balanced heterodyne detection with application to 2D IR spectroscopy.

    PubMed

    Middleton, Chris T; Strasfeld, David B; Zanni, Martin T

    2009-08-17

    We demonstrate amplitude, phase and polarization shaping of femtosecond mid-IR pulses using a germanium acousto-optical modulator by independently shaping the frequency-dependent amplitudes and phases of two orthogonally polarized pulses which are then collinearly overlapped using a wire-grid polarizer. We use a feedback loop to set and stabilize the relative phase of the orthogonal pulses. We have also used a wire-grid polarizer to implement polarization-based balanced heterodyne detection for improved signal-to-noise of 2D IR spectra collected in a pump-probe geometry. Applications include coherent control of molecular vibrations and improvements in multidimensional IR spectroscopy.

  9. Investigation of the nitrogen hyperfine coupling of the second stable radical in γ-irradiated L-alanine crystals by 2D-HYSCORE spectroscopy

    NASA Astrophysics Data System (ADS)

    Maltar-Strmečki, Nadica; Rakvin, Boris

    2012-09-01

    The second stable radical, NH3+C(CH3)COO, R2, in the γ-irradiated single crystal of L-alanine and its fully 15N-enriched analogue were studied by an advanced pulsed EPR technique, 2D-HYSCORE (two-dimensional hyperfine sublevel correlation) spectroscopy at 200 K. The nitrogen hyperfine coupling tensor of the R2 radical was determined from the HYSCORE data and provides new experimental data for improved characterization of the R2 radical in the crystal lattice. The results obtained complement the experimental proton data available for the R2 radical and could lead to increased accuracy and reliability of EPR spectrum simulations.

  10. Front face fluorescence spectroscopy and visible spectroscopy coupled with chemometrics have the potential to characterise ripening of Cabernet Franc grapes.

    PubMed

    Le Moigne, Marine; Dufour, Eric; Bertrand, Dominique; Maury, Chantal; Seraphin, Denis; Jourjon, Frédérique

    2008-07-21

    The potential of front-face spectroscopy for grape ripening dates discrimination was investigated on Cabernet Franc grapes from three parcels located on the Loire Valley and for six ripening dates. The 18 batches were analysed by front-face fluorescence spectroscopy and visible spectroscopy. The excitation spectra (250-310nm, emission wavelength=350nm) were characterised by a shoulder at 280nm. Grapes spectra were classified by factorial discriminant analysis (FDA). Ripening dates were well predicted by fluorescence spectra: grapes before veraison were separated from grapes after veraison and almost every ripening date was identified. The common spectroscopic space obtained by CCSWA showed that wavelengths corresponding to anthocyanin absorption in the visible were correlated to fluorescence wavelengths around the starting and ending points of the shoulder (263 and at 292nm). Then, regression models were investigated to predict total soluble solids (TSS), total acidity, malvidin-3G, total anthocyanins and total phenolics content from visible and fluorescence spectra. To predict technological indicators (TSS and total acidity), the PLS model with visible spectra (RMSECV=0.82 degrees Brix or 0.96gL(-1) H(2)SO(4)) was better than those with fluorescence one (RMSECV=1.39 degrees Brix or 2.06gL(-1) H(2)SO(4)). For malvidin-3G and total anthocyanins, all R(c)(2) and R(cv)(2) were superior to 0.90 and RMSECV were low. Visible and fluorescence spectroscopies succeeded in predicting anthocyanin content. Concerning total phenolic, the best prediction was provided by fluorescence spectroscopy.

  11. Endogenous synchronous fluorescence spectroscopy (SFS) of basal cell carcinoma-initial study

    NASA Astrophysics Data System (ADS)

    Borisova, E.; Zhelyazkova, Al.; Keremedchiev, M.; Penkov, N.; Semyachkina-Glushkovskaya, O.; Avramov, L.

    2016-01-01

    The human skin is a complex, multilayered and inhomogeneous organ with spatially varying optical properties. Analysis of cutaneous fluorescence spectra could be a very complicated task; therefore researchers apply complex mathematical tools for data evaluation, or try to find some specific approaches, that would simplify the spectral analysis. Synchronous fluorescence spectroscopy (SFS) allows improving the spectral resolution, which could be useful for the biological tissue fluorescence characterization and could increase the tumour detection diagnostic accuracy.

  12. Surface Plasmon Resonances in 1D and 2D Arrays of Metal Nanoparticles for the Control of Enhanced Spectroscopies

    DTIC Science & Technology

    2011-01-24

    currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM-YYYY) 2 . REPORT TYPE 3. DATES...SPECTROSCOPIES FA9550-09-1-0579 Noguez, Cecilia Roman-Velazquez, Carlos E. Angulo, Ali M. Instituto de Fisica Universidad Nacional Autonoma de Mexico...representation, nanoshells, nanospheres U U U SAR 2 Cecilia Noguez +52 (55) 5622 5106 Final Technical Report Grant/Contract Title: SURFACE PLASMON

  13. A fluorescence spectroscopy study of traditional Chinese medicine Angelica

    NASA Astrophysics Data System (ADS)

    Zhao, Hongyan; Song, Feng; Liu, Shujing; Chen, Guiyang; Wei, Chen; Liu, Yanling; Liu, Jiadong

    2013-10-01

    By measuring the fluorescence spectra of Chinese medicine (CM) Angelica water solutions with different concentrations from 0.025 to 2.5 mg/mL, results showed that the fluorescence intensity was proportional to the concentration. Through fluorescence spectra of Angelica solution under different pH values, results indicated coumarin compounds were the active ingredients of Angelica. We also observed fluorescence quenching of the Angelica solution in the presence of spherical silver nanoparticles with radius of 12 nm. Keeping a certain value for the volume of the silver nanoparticles, the fluorescence intensity at 402 nm was linearly proportional to the Angelica in the range of 1-3 mg/mL.

  14. Single gold nanoparticles to enhance the detection of single fluorescent molecules at micromolar concentration using fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Punj, Deep; Rigneault, Hervé; Wenger, Jérôme

    2014-05-01

    Single nanoparticles made of noble metals are strongly appealing to develop practical applications to detect fluorescent molecules in solution. Here, we detail the use of a single gold nanoparticle of 100 nm diameter to enhance the detection of single Alex Fluor 647 fluorescent molecules at high concentrations of several micromolar. We discuss the implementation of fluorescence correlation spectroscopy, and provide a new method to reliably extract the enhanced fluorescence signal stemming from the nanoparticle near-field from the background generated in the confocal volume. The applicability of our method is checked by reporting the invariance of the single molecule results as function of the molecular concentration, and the experimental data is found in good agreement with numerical simulations.

  15. Evaluation on intrinsic quality of licorice influenced by environmental factors by using FTIR combined with 2D-IR correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhou, Ying-qun; Yu, Hua; Zhang, Yan-ling; Sun, Su-qin; Chen, Shi-lin; Zhao, Run-huai; Zhou, Qun; Noda, Isao

    2010-06-01

    To evaluate the intrinsic quality of licorice influenced by environmental factors, the spectral comparison of licorice from two typical ecological habitats was conducted by using FTIR and 2D-IR correlation spectroscopy. There were differences in the peak intensities of 1155, 1076 and 1048 cm -1 of FTIR profiles. The difference was amplified by the second derivative spectrum for the peak intensities at 1370, 1365 and 1317 cm -1 and the peak shape in 958-920 cm -1 and 1050-988 cm -1. The synchronous 2D-IR spectra within the range of 860-1300 cm -1 were classified into type I and type II and their frequency in the two groups was noticeably different. Although the chemical compounds of licorice samples from two areas were generally similar, the contents of starch, calcium oxalate, and some chemical compounds containing alcohol hydroxyl group were different, indicating the influence of precipitation and temperature. This study demonstrates that the systematical analysis of FTIR, the second derivative spectrum and 2D-IR can effectively determine the differences in licorice samples from different ecological habitats.

  16. [Rapid identification of potato cultivars using NIR-excited fluorescence and Raman spectroscopy].

    PubMed

    Dai, Fen; Bergholt, Mads Sylvest; Benjamin, Arnold Julian Vinoj; Hong, Tian-Sheng; Zhiwei, Huang

    2014-03-01

    Potato is one of the most important food in the world. Rapid and noninvasive identification of potato cultivars plays a important role in the better use of varieties. In this study, The identification ability of optical spectroscopy techniques, including near-infrared (NIR) Raman spectroscopy and NIR fluorescence spectroscopy, for invasive detection of potato cultivars was evaluated. A rapid NIR Raman spectroscopy system was applied to measure the composite Raman and NIR fluorescence spectroscopy of 3 different species of potatoes (98 samples in total) under 785 nm laser light excitation. Then pure Raman and NIR fluorescence spectroscopy were abstracted from the composite spectroscopy, respectively. At last, the partial least squares-discriminant analysis (PLS-DA) was utilized to analyze and classify Raman spectra of 3 different types of potatoes. All the samples were divided into two sets at random: the calibration set (74samples) and prediction set (24 samples), the model was validated using a leave-one-out, cross-validation method. The results showed that both the NIR-excited fluorescence spectra and pure Raman spectra could be used to identify three cultivars of potatoes. The fluorescence spectrum could distinguish the Favorita variety well (sensitivity: 1, specificity: 0.86 and accuracy: 0.92), but the result for Diamant (sensitivity: 0.75, specificity: 0.75 and accuracy: 0. 75) and Granola (sensitivity: 0.16, specificity: 0.89 and accuracy: 0.71) cultivars identification were a bit poorer. We demonstrated that Raman spectroscopy uncovered the main biochemical compositions contained in potato species, and provided a better classification sensitivity, specificity and accuracy (sensitivity: 1, specificity: 1 and accuracy: 1 for all 3 potato cultivars identification) among the three types of potatoes as compared to fluorescence spectroscopy.

  17. Fluorescence spectroscopy of the retina from scrapie-infected mice.

    PubMed

    Bose, Sayantan; Schönenbrücher, Holger; Richt, Jürgen A; Casey, Thomas A; Rasmussen, Mark A; Kehrli, Marcus E; Petrich, Jacob W

    2013-01-01

    Recently, we have proposed that the fluorescence spectra of sheep retina can be well correlated with the presence or absence of scrapie. Scrapie is the most widespread TSE (transmissible spongiform encephalopathy) affecting sheep and goats worldwide. Mice eyes have been previously reported as a model system to study age-related accumulation of lipofuscin, which has been investigated by monitoring the increasing fluorescence with age covering its entire life span. The current work aims at developing mice retina as a convenient model system to diagnose scrapie and other fatal TSE diseases in animals such as sheep and cows. The objective of the research reported here was to determine whether the spectral features are conserved between two different species namely mice and sheep, and whether an appropriate small animal model system could be identified for diagnosis of scrapie based on the fluorescence intensity in retina. The results were consistent with the previous reports on fluorescence studies of healthy and scrapie-infected retina of sheep. The fluorescence from the retinas of scrapie-infected sheep was significantly more intense and showed more heterogeneity than that from the retinas of uninfected mice. Although the structural characteristics of fluorescence spectra of scrapie-infected sheep and mice eyes are slightly different, more importantly, murine retinas reflect the enhancement of fluorescence intensity upon infecting the mice with scrapie, which is consistent with the observations in sheep eyes.

  18. Direct solid surface fluorescence spectroscopy of standard chemicals and humic acid in ternary system.

    PubMed

    Mounier, S; Nicolodelli, G; Redon, R; Milori, D M B P

    2017-04-15

    The front face fluorescence spectroscopy is often used to quantify chemicals in well-known matrices as it is a rapid and powerful technique, with no sample preparation. However it was not used to investigate extracted organic matter like humic substances. This work aims to fully investigate for the first time front face fluorescence spectroscopy response of a ternary system including boric acid, tryptophan and humic substances, and two binaries system containing quinine sulfate or humic substance in boric acid. Pure chemicals, boric acid, tryptophan, quinine sulfate and humic acid were mixed together in solid pellet at different contents from 0 to 100% in mass. The measurement of excitation emission matrix of fluorescence (3D fluorescence) and laser induced fluorescence were then done in the front face mode. Fluorescence matrices were decomposed using the CP/PARAFAC tools after scattering treatments. Results show that for 3D fluorescence there is no specific component for tryptophan and quinine sulfate, and that humic substances lead to a strong extinction effect for mixture containing quinine sulfate. Laser induced fluorescence gives a very good but non-specific related response for both quinine sulfate and tryptophan. No humic substances fluorescence response was found, but extinction effect is observed as for 3D fluorescence. This effect is stronger for quinine sulfate than for tryptophan. These responses were modeled using a simple absorbance versus emission model.

  19. Direct solid surface fluorescence spectroscopy of standard chemicals and humic acid in ternary system

    NASA Astrophysics Data System (ADS)

    Mounier, S.; Nicolodelli, G.; Redon, R.; Milori, D. M. B. P.

    2017-04-01

    The front face fluorescence spectroscopy is often used to quantify chemicals in well-known matrices as it is a rapid and powerful technique, with no sample preparation. However it was not used to investigate extracted organic matter like humic substances. This work aims to fully investigate for the first time front face fluorescence spectroscopy response of a ternary system including boric acid, tryptophan and humic substances, and two binaries system containing quinine sulfate or humic substance in boric acid. Pure chemicals, boric acid, tryptophan, quinine sulfate and humic acid were mixed together in solid pellet at different contents from 0 to 100% in mass. The measurement of excitation emission matrix of fluorescence (3D fluorescence) and laser induced fluorescence were then done in the front face mode. Fluorescence matrices were decomposed using the CP/PARAFAC tools after scattering treatments. Results show that for 3D fluorescence there is no specific component for tryptophan and quinine sulfate, and that humic substances lead to a strong extinction effect for mixture containing quinine sulfate. Laser induced fluorescence gives a very good but non-specific related response for both quinine sulfate and tryptophan. No humic substances fluorescence response was found, but extinction effect is observed as for 3D fluorescence. This effect is stronger for quinine sulfate than for tryptophan. These responses were modeled using a simple absorbance versus emission model.

  20. Laser-induced fluorescence spectroscopy of the secondary cataract

    NASA Astrophysics Data System (ADS)

    Maslov, N. A.; Larionov, P. M.; Rozhin, I. A.; Druzhinin, I. B.; Chernykh, V. V.

    2016-06-01

    Excitation-emission matrices of laser-induced fluorescence of lens capsule epithelium, the lens nucleus, and the lens capsule are investigated. A solid-state laser in combination with an optical parametric generator tunable in the range from 210 to 350 nm was used for excitation of fluorescence. The spectra of fluorescence of all three types of tissues exhibit typical features that are specific to them and drastically differ from one another. This effect can be used for intrasurgical control of presence of residual lens capsule epithelium cells in the capsular bag after surgical treatment of a cataract.

  1. Detection of mechanical and disease stresses in citrus plants by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Belasque, J., Jr.; Gasparoto, M. C. G.; Marcassa, L. G.

    2008-04-01

    We have investigated the detection of mechanical and disease stresses in citrus plants (Citrus limonia [L.] Osbeck) using laser-induced fluorescence spectroscopy. Due to its economic importance we have chosen to investigate the citrus canker disease, which is caused by the Xanthomonas axonopodis pv. citri bacteria. Mechanical stress was also studied because it plays an important role in the plant's infection by such bacteria. A laser-induced fluorescence spectroscopy system, composed of a spectrometer and a 532 nm10 mW excitation laser was used to perform fluorescence spectroscopy. The ratio of two chlorophyll fluorescence bands allows us to detect and discriminate between mechanical and disease stresses. This ability to discriminate may have an important application in the field to detect citrus canker infected trees.

  2. Two Keggin-type heteropolytungstates with transition metal as a central atom: Crystal structure and magnetic study with 2D-IR correlation spectroscopy

    SciTech Connect

    Chai, Feng; Chen, YiPing; You, ZhuChai; Xia, ZeMin; Ge, SuZhi; Sun, YanQiong; Huang, BiHua

    2013-06-01

    Two Keggin-type heteropolytungstates, [Co(phen)₃]₃[CoW₁₂O₄₀]·9H₂O 1 (phen=1,10-phenanthroline) and [Fe(phen)₃]₂[FeW₁₂O₄₀]·H₃O·H₂O 2, have been synthesized via the hydrothermal technique and characterized by single crystal X-ray diffraction analyses, IR, XPS, TG analysis, UV–DRS, XRD, thermal-dependent and magnetic-dependent 2D-COS IR (two-dimensional infrared correlation spectroscopy). Crystal structure analysis reveals that the polyanions in compound 1 are linked into 3D supramolecule through hydrogen bonding interactions between lattice water molecules and terminal oxygen atoms of polyanion units, and [Co(phen)₃]²⁺ cations distributed in the polyanion framework with many hydrogen bonding interactions. The XPS spectra indicate that all the Co atoms in 1 are +2 oxidation state, the Fe atoms in 2 existing with +2 and +3 mixed oxidation states. - Graphical abstract: The magnetic-dependent synchronous 2D correlation IR spectra of 1 (a), 2 (b) over 0–50 mT in the range of 600–1000 cm⁻¹, the obvious response indicate two Keggin polyanions skeleton susceptible to applied magnetic field. Highlights: • Two Keggin-type heteropolytungstates with transition metal as a central atom has been obtained. • Compound 1 forms into 3D supramolecular architecture through hydrogen bonding between water molecules and polyanions. • Magnetic-dependent 2D-IR correlation spectroscopy was introduced to discuss the magnetism of polyoxometalate.

  3. Excitation emission and time-resolved fluorescence spectroscopy of selected varnishes used in historical musical instruments.

    PubMed

    Nevin, Austin; Echard, Jean-Philippe; Thoury, Mathieu; Comelli, Daniela; Valentini, Gianluca; Cubeddu, Rinaldo

    2009-11-15

    The analysis of various varnishes from different origins, which are commonly found on historical musical instruments was carried out for the first time with both fluorescence excitation emission spectroscopy and laser-induced time-resolved fluorescence spectroscopy. Samples studied include varnishes prepared using shellac, and selected diterpenoid and triterpenoid resins from plants, and mixtures of these materials. Fluorescence excitation emission spectra have been collected from films of naturally aged varnishes. In parallel, time-resolved fluorescence spectroscopy of varnishes provides means for discriminating between short- (less than 2.0 ns) and long-lived (greater than 7.5 ns) fluorescence emissions in each of these complex materials. Results suggest that complementary use of the two non destructive techniques allows a better understanding of the main fluorophores responsible for the emission in shellac, and further provides means for distinguishing the main classes of other varnishes based on differences in fluorescence lifetime behaviour. Spectrofluorimetric data and time resolved spectra presented here may form the basis for the interpretation of results from future in situ fluorescence examination and time resolved fluorescence imaging of varnished musical instruments.

  4. Two-photon fluorescence correlation spectroscopy with high count rates and low background using dielectric microspheres

    PubMed Central

    Aouani, Heykel; Schön, Peter; Brasselet, Sophie; Rigneault, Hervé; Wenger, Jérôme

    2010-01-01

    Two-photon excitation fluorescence is a powerful technique commonly used for biological imaging. However, the low absorption cross section of this non-linear process is a critical issue for performing biomolecular spectroscopy at the single molecule level. Enhancing the two-photon fluorescence signal would greatly improve the effectiveness of this technique, yet current methods struggle with medium enhancement factors and/or high background noise. Here, we show that the two-photon fluorescence signal from single Alexa Fluor 488 molecules can be enhanced up to 10 times by using a 3 µm diameter latex sphere while adding almost no photoluminescence background. We report a full characterization of the two-photon fluorescence enhancement by a single microsphere using fluorescence correlation spectroscopy. This opens new routes to enhance non-linear optical signals and extend biophotonic applications. PMID:21258531

  5. Effect of solvent polarity on the vibrational dephasing dynamics of the nitrosyl stretch in an Fe(II) complex revealed by 2D IR spectroscopy.

    PubMed

    Brookes, Jennifer F; Slenkamp, Karla M; Lynch, Michael S; Khalil, Munira

    2013-07-25

    The vibrational dephasing dynamics of the nitrosyl stretching vibration (ν(NO)) in sodium nitroprusside (SNP, Na2[Fe(CN)5NO]·2H2O) are investigated using two-dimensional infrared (2D IR) spectroscopy. The ν(NO) in SNP acts as a model system for the nitrosyl ligand found in metalloproteins which play an important role in the transportation and detection of nitric oxide (NO) in biological systems. We perform a 2D IR line shape study of the ν(NO) in the following solvents: water, deuterium oxide, methanol, ethanol, ethylene glycol, formamide, and dimethyl sulfoxide. The frequency of the ν(NO) exhibits a large vibrational solvatochromic shift of 52 cm(-1), ranging from 1884 cm(-1) in dimethyl sulfoxide to 1936 cm(-1) in water. The vibrational anharmonicity of the ν(NO) varies from 21 to 28 cm(-1) in the solvents used in this study. The frequency-frequency correlation functions (FFCFs) of the ν(NO) in SNP in each of the seven solvents are obtained by fitting the experimentally obtained 2D IR spectra using nonlinear response theory. The fits to the 2D IR line shape reveal that the spectral diffusion time scale of the ν(NO) in SNP varies from 0.8 to 4 ps and is negatively correlated with the empirical solvent polarity scales. We compare our results with the experimentally determined FFCFs of other charged vibrational probes in polar solvents and in the active sites of heme proteins. Our results suggest that the vibrational dephasing dynamics of the ν(NO) in SNP reflect the fluctuations of the nonhomogeneous electric field created by the polar solvents around the nitrosyl and cyanide ligands. The solute solvent interactions occurring at the trans-CN ligand are sensed through the π-back-bonding network along the Fe-NO bond in SNP.

  6. Ultrafast Fluorescence Spectroscopy via Upconversion: Applications to Biophysics

    PubMed Central

    Xu, Jianhua; Knutson, Jay R.

    2012-01-01

    This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss applications in biophysics, particularly the measurement of time-resolved fluorescence spectra of proteins (with subpicosecond time resolution). Application of this technique to biophysical problems such as dynamics of tryptophan, peptides, proteins, and nucleic acids is reviewed. PMID:19152860

  7. Fluorescence spectroscopy of gastrointestinal tumors using δ-ALA

    NASA Astrophysics Data System (ADS)

    Borisova, E. G.; Vladimirov, B. G.; Angelov, I. G.; Avramov, L. A.

    2007-03-01

    In the recent study delta-aminolevulinic acid/Protoporphyrin IX (δ-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus and stomach. The δ-ALA is administered per os six hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built to use the light guide of standard video-endoscopic system (Olimpus Corp.). Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer (USB4000, OceanOptics Inc.). The fluorescence detected from tumor sites has very complex spectral origins. It consists of autofluorescence, fluorescence from exogenous fluorophores and re-absorption from the chromophores accumulated in the tissue investigated. Mucosa autofluorescence lies at 450-600 nm region. The fluorescence of PpIX is clearly pronounced at the 630-710 nm region. Deep minima in the tumor fluorescence signals are observed in the region 540-575 nm, related to hemoglobin re-absorption. Such high hemoglobin content is an indication of the tumors neovascularisation and it is clearly pronounced in all dysplastic and tumor sites investigated. The lack of fluorescence peaks in the red spectral area for normal mucosa is an indication for selective accumulation of δ-ALA/PpIX only in abnormal sites and gives high contrast when lesion borders are determined from clinicians during video observation in the process of diagnostic procedure. Very good correlation between fluorescence signals and histology examination results of the lesions investigated is achieved.

  8. Solvation of fluoro-acetonitrile in water by 2D-IR spectroscopy: A combined experimental-computational study

    SciTech Connect

    Cazade, Pierre-André; Das, Akshaya K.; Tran, Halina; Kläsi, Felix; Hamm, Peter; Bereau, Tristan; Meuwly, Markus

    2015-06-07

    The solvent dynamics around fluorinated acetonitrile is characterized by 2-dimensional infrared spectroscopy and atomistic simulations. The lineshape of the linear infrared spectrum is better captured by semiempirical (density functional tight binding) mixed quantum mechanical/molecular mechanics simulations, whereas force field simulations with multipolar interactions yield lineshapes that are significantly too narrow. For the solvent dynamics, a relatively slow time scale of 2 ps is found from the experiments and supported by the mixed quantum mechanical/molecular mechanics simulations. With multipolar force fields fitted to the available thermodynamical data, the time scale is considerably faster—on the 0.5 ps time scale. The simulations provide evidence for a well established CF–HOH hydrogen bond (population of 25%) which is found from the radial distribution function g(r) from both, force field and quantum mechanics/molecular mechanics simulations.

  9. Fluorescence spectroscopy of anisole at elevated temperatures and pressures

    NASA Astrophysics Data System (ADS)

    Tran, K. H.; Morin, C.; Kühni, M.; Guibert, P.

    2014-06-01

    Laser-induced fluorescence of anisole as tracer of isooctane at an excitation wavelength of 266 nm was investigated for conditions relevant to rapid compression machine studies and for more general application of internal combustion engines regarding temperature, pressure, and ambient gas composition. An optically accessible high pressure and high temperature chamber was operated by using different ambient gases (Ar, N2, CO2, air, and gas mixtures). Fluorescence experiments were investigated at a large range of pressure and temperature (0.2-4 MPa and 473-823 K). Anisole fluorescence quantum yield decreases strongly with temperature for every considered ambient gas, due to efficient radiative mechanisms of intersystem crossing. Concerning the pressure effect, the fluorescence signal decreases with increasing pressure, because increasing the collisional rate leads to more important non-radiative collisional relaxation. The quenching effect is strongly efficient in oxygen, with a fluorescence evolution described by Stern-Volmer relation. The dependence of anisole fluorescence versus thermodynamic parameters suggests the use of this tracer for temperature imaging in specific conditions detailed in this paper. The calibration procedure for temperature measurements is established for the single-excitation wavelength and two-color detection technique.

  10. Comparing Compositions of Modern Cast Bronze Sculptures: Optical Emission Spectroscopy Versus x-Ray Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Young, M. L.; Dunand, D. C.

    2015-07-01

    Bulk elemental compositions of 74 modern cast bronze sculptures from the collection at the Art Institute of Chicago, the Philadelphia Museum of Art, and the Rodin Museum (Philadelphia, PA) were determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and a handheld x-ray fluorescence (XRF) spectrometer. The elemental compositions of the cast sculptures as measured previously by ICP-OES and presently by XRF are compared: A good match is found between the two methods for the base metal (Cu) and the two majority alloying elements (Zn and Sn). For both ICP-OES and XRF data, when the Zn composition is plotted versus the Sn composition, three discernable clusters are found that are related to the artist, foundry, casting date, and casting method; they consist of (A) high-zinc brass, (B) low-zinc, low-tin brass, and (C) low-zinc, tin bronze. Thus, our study confirms that the relatively fast, nondestructive XRF spectrometry can be used effectively over slower and invasive, but more accurate, ICP-OES to help determine a sculpture's artist, foundry, date of creation, date of casting, and casting method.

  11. Intracellular distribution of fluorescent copper and zinc bis(thiosemicarbazonato) complexes measured with fluorescence lifetime spectroscopy.

    PubMed

    Hickey, James L; James, Janine L; Henderson, Clare A; Price, Katherine A; Mot, Alexandra I; Buncic, Gojko; Crouch, Peter J; White, Jonathan M; White, Anthony R; Smith, Trevor A; Donnelly, Paul S

    2015-10-05

    The intracellular distribution of fluorescently labeled copper and zinc bis(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis(thiosemicarbazonato) complex known as Cu(II)(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu(II) and Zn(II) complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper(II)bis(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu(II)(atsm) in models of amyotrophic lateral sclerosis and Parkinson's disease.

  12. Real-time observation of multiexcitonic states in ultrafast singlet fission using coherent 2D electronic spectroscopy.

    PubMed

    Bakulin, Artem A; Morgan, Sarah E; Kehoe, Tom B; Wilson, Mark W B; Chin, Alex W; Zigmantas, Donatas; Egorova, Dassia; Rao, Akshay

    2016-01-01

    Singlet fission is the spin-allowed conversion of a spin-singlet exciton into a pair of spin-triplet excitons residing on neighbouring molecules. To rationalize this phenomenon, a multiexcitonic spin-zero triplet-pair state has been hypothesized as an intermediate in singlet fission. However, the nature of the intermediate states and the underlying mechanism of ultrafast fission have not been elucidated experimentally. Here, we study a series of pentacene derivatives using ultrafast two-dimensional electronic spectroscopy and unravel the origin of the states involved in fission. Our data reveal the crucial role of vibrational degrees of freedom coupled to electronic excitations that facilitate the mixing of multiexcitonic states with singlet excitons. The resulting manifold of vibronic states drives sub-100 fs fission with unity efficiency. Our results provide a framework for understanding singlet fission and show how the formation of vibronic manifolds with a high density of states facilitates fast and efficient electronic processes in molecular systems.

  13. Hybrid native phosphorescence and fluorescence spectroscopy for cancer detection

    NASA Astrophysics Data System (ADS)

    Alimova, Alexandra; Katz, A.; Sriramoju, Vidyasagar; Budansky, Yuri; Bykov, Alexei A.; Zeylikovich, Roman; Alfano, R. R.

    2006-02-01

    Native fluorescence of tissues in the UV and visible spectral regions has been investigated for over two decades. Native fluorescence has been demonstrated to be an accurate tools for distinguish normal tissue from malignant and pre-malignant. Prior investigations have demonstrated that there are several ratio-based algorithms, which can distinguish malignant tissue from normal with high sensitivity and specificity.1 The wavelength combinations used in these ratios isolate the contributions from pairs of tissue fluorophors, one of which is frequently tryptophan (trp), the predominant tissue fluorophore with excitation in the UV (250-300 nm). In this work, algorithms using a combination of native fluorescence and trp phosphorescence were developed which show promise for providing enhanced detection accuracy. Using optical fibers to collect the emission from the specimen allowed interrogation of small regions of tissue, providing precise spatial information. Using a specially designed setup, specimens were excited in the UV and spectra were collected in the range of 300 to 700 nm. Three main emission bands were selected for analysis: 340 nm (trp fluorescence); 420 - 460 nm band (fluorescence from the extra cellular matrix); and 500 - 520 nm (trp phosphorescence). Normal specimens consistently exhibited a low ratio (<10) of 345 to 500 nm emission intensity while this same ratio was consistently high (>15) for cancer specimens. Creating intensities ratio maps from the tissue allows one to localize the malignant regions with high spatial precision. The study was performed on ex vivo human breast tissues. The ratio analysis correlated well with histopathology.

  14. Research of the interaction between kangai injection and human serum albumin by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Ye, Changbin; Lin, Xiaogang; Zhu, Hao; Li, Wenchao; Wu, Jie

    2015-10-01

    The interaction between drugs and serum albumin is the theoretical basis of pharmacology research. Kangai injection with invigorating Qi, enhancing the immune function, is widely used for a variety of malignant tumor treatment. Fluorescence spectroscopy was adopted due to its high sensitivity and other advantages. The interaction between kangai injection and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence spectroscopy and UV-Vis absorption spectroscopy. The results of fluorescence spectrum at three temperature (296K, 303K and 310K) showed the degree of binding at 310K is the highest. Also, the maximum emission peak has a slight blue shift, which indicates that the interaction between kangai injection and HSA has an effect on the conformation of HSA. That is, the microenvironment of tryptophan increase hydrophobic due to the increase of the concentration of kangai injection. Results obtained from analysis of fluorescence spectrum and fluorescence intensity indicated that kangai injection has a strong ability to quench the intrinsic fluorescence of HSA. And according to the Stern-Volume equation, the quenching mechanism is static quenching, which is further proved by the UV-Vis absorption spectroscopy.

  15. Time-resolved Hyperspectral Fluorescence Spectroscopy using Frequency Modulated Excitation

    SciTech Connect

    ,; Neill, M

    2012-07-01

    An intensity-modulated excitation light source is used together with a micro channel plate intensified CCD (ICCD) detector gated at a slightly different frequency to generate a beat frequency from a fluorescent sample. The addition of a spectrograph produces a hyperspectral time-resolved data product where the resulting beat frequency is detected with a low frame rate camera. Measuring the beat frequency of the spectrum as a function of time allows separation of the excited fluorescence from ambient constant light sources. The excitation and detector repetition rates are varied over a range of discrete frequencies, and the phase shift of the beat wave maps out the emission decay rate(s).

  16. Synthesizing and Characterizing Graphene via Raman Spectroscopy: An Upper-Level Undergraduate Experiment That Exposes Students to Raman Spectroscopy and a 2D Nanomaterial

    ERIC Educational Resources Information Center

    Parobek, David; Shenoy, Ganesh; Zhou, Feng; Peng, Zhenbo; Ward, Michelle; Liu, Haitao

    2016-01-01

    In this upper-level undergraduate experiment, students utilize micro-Raman spectroscopy to characterize graphene prepared by mechanical exfoliation and chemical vapor deposition (CVD). The mechanically exfoliated samples are prepared by the students while CVD graphene can be purchased or obtained through outside sources. Owing to the intense Raman…

  17. Quantitative analysis of essential oils of Thymus daenensis using laser-induced fluorescence and Raman spectroscopy.

    PubMed

    Khoshroo, H; Khadem, H; Bahreini, M; Tavassoli, S H; Hadian, J

    2015-11-10

    Laser-induced fluorescence and Raman spectroscopy are used for the investigation of different genotypes of Thymus daenensis native to the Ilam province of Iran. Different genotypes of T. daenensis essential oils, labeled T1 through T7, possess slight differences with regard to the composition of the thymol. The gas chromatography-mass spectrometry (GC-MS) method is performed to determine the concentration of each constituent as a reference method. The Raman spectra of different concentrations of pure thymol dissolved in hexane as standard samples are obtained via a laboratory prototype Raman spectroscopy setup for the calculation of the calibration curve. The regression coefficient and limit of detection are calculated. The possibility of the differentiation of different genotypes of T. daenensis is also examined by laser-induced fluorescence spectroscopy, although we do not know the exact amounts of their components. All the fluorescence spectral information is used jointly by cluster analysis to differentiate between 7 genotypes. Our results demonstrate the acceptable precision of Raman spectroscopy with GC-MS and corroborate the capacity of Raman spectroscopy in applications in the quantitative analysis field. Furthermore, the cluster analysis results show that laser-induced fluorescence spectroscopy is an acceptable technique for the rapid classification of different genotypes of T. daenensis without having any previous information of their exact amount of constituents. So, the ability to rapidly and nondestructively differentiate between genotypes makes it possible to efficiently select high-quality herbs from many samples.

  18. Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.

    PubMed

    Rezgui, Rachid; Blumer, Katy; Yeoh-Tan, Gilbert; Trexler, Adam J; Magzoub, Mazin

    2016-07-01

    Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane.

  19. The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors.

    PubMed

    Kilpatrick, Laura E; Hill, Stephen J

    2016-04-15

    The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand-receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties.

  20. Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine.

    PubMed

    Boreham, Alexander; Brodwolf, Robert; Walker, Karolina; Haag, Rainer; Alexiev, Ulrike

    2016-12-24

    The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM) for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

  1. The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors

    PubMed Central

    Kilpatrick, Laura E.; Hill, Stephen J.

    2016-01-01

    The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties. PMID:27068980

  2. Fluorescence spectroscopy of the retina from scrapie-infected mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, we have proposed that the fluorescence spectra of sheep retina can be well correlated to the presence or absence of scrapie. Scrapie is the most widespread TSE (transmissible spongiform encephalopathy) affecting sheep and goats worldwide. Mice eyes have been previously reported as a model ...

  3. Two-photon fluorescence excitation spectroscopy of biological molecules

    NASA Astrophysics Data System (ADS)

    Meshalkin, Yuri P.; Alfimov, E. E.; Groshev, D. E.; Makukha, V. K.

    1996-06-01

    The UV fluorescence spectra of aromatic amino-acids and some proteins at two photon excitation by second harmonic of Nd:YAG laser are received. Two-photon absorption cross sections of tryptophan, tyrosine, phenylalanine and proteins: bovine serum albumin, lysozyme, trypsin, (alpha) - chymotrypsinogen and pepsin at wavelength 532 nm were measured by means of the two-quantum standard method.

  4. Fluorescence Spectroscopy of Conformational Changes of Single LH2 Complexes

    PubMed Central

    Rutkauskas, Danielis; Novoderezhkin, Vladimir; Cogdell, Richard J.; van Grondelle, Rienk

    2005-01-01

    We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between long-lived quasi-stable levels differing by up to 30 nm. The frequency and size of these fluorescence peak movements were found to increase linearly with the excitation intensity. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The probability for a particle to undergo significant spectral shift in either direction was found to be roughly the same. Using the modified Redfield theory, the observed changes in spectral shape and intensity were accounted for by changes in the realization of the static disorder. Long lifetimes of the quasi-stable states suggest large energetic barriers between the states characterized by different emission spectra. PMID:15501944

  5. Variation of fluorescence spectroscopy during the menstrual cycle

    NASA Astrophysics Data System (ADS)

    Macaulay, Calum; Richards-Kortum, Rebecca; Utzinger, Urs; Fedyk, Amanda; Neely Atkinson, E.; Cox, Dennis; Follen, Michele

    2002-06-01

    Cervical autofluorescence has been demonstrated to have potential for real-time diagnosis. Inter-patient and intra-patient variations in fluorescence intensity have been measured. Inter-patient measurements may vary by a factor of ten, while intra-patient measurements may vary by a factor of two. Age and menopausal status have been demonstrated to account for some of the variations, while race and smoking have not. In order to explore in detail the role of the menstrual cycle in intra-patient variation, a study was designed to measure fluorescence excitation emission matrices (EEMs) in patients daily throughout one cycle. Ten patients with a history of normal menstrual cycles and normal Papanicolaou smears underwent daily measurements of fluorescence EEMs from three colposcopically normal sites throughout one menstrual cycle. Changes in signals from porphyrin, NADH, and FAD fluorescence and blood absorption were noted when the data was viewed in a graphical format. Visually interpreted features of the EEMs in this graphical format did not appear to correlate with the day of the menstrual cycle with the exception that blood absorption features were more prominent during the menstrual phase (during which bleeding occurs), suggesting that measurements during the menstrual phase should be avoided. Variations in cycle date likely do not account for inter- or intra-patient variations.

  6. Potential of fluorescence spectroscopy to predict fatty acid composition of beef.

    PubMed

    Aït-Kaddour, A; Thomas, A; Mardon, J; Jacquot, S; Ferlay, A; Gruffat, D

    2016-03-01

    The present study aimed to evaluate and compare the ability of front face (FFFS) and synchronous fluorescence spectroscopy (SFS) to predict total fat and FA composition of beef LT muscles coming from 36 animals of 3 breeds (Angus, Limousin and Blond d'Aquitaine). The regression models were performed by using Partial Least Square (PLS) method. In spite of the low number of samples used, the results of this preliminary study demonstrated the ability of fluorescence spectroscopy to predict meat lipids. Nonetheless, the results suggested that the fluorescence spectroscopy is more suited to measure SFA (R(2)p≥0.66; RPD≥2.29) and MUFA (R(2)p≥0.48; RPD≥1.49) than PUFA (R(2)p≤0.48; RPD≤1.63). Moreover, R(2) and RPD factors obtained with FFFS were greater compared to the ones obtained with SFS suggesting that FFFS is more adapted to measure lipid composition of beef meat.

  7. 2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

    NASA Astrophysics Data System (ADS)

    Ghosh, Ayanjeet; Wang, Jun; Moroz, Yurii S.; Korendovych, Ivan V.; Zanni, Martin; DeGrado, William F.; Gai, Feng; Hochstrasser, Robin M.

    2014-06-01

    Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs.

  8. Synthesis, structure and temperature-depended 2D IR correlation spectroscopy of an organo-bismuth benzoate with 1,10-phenanthroline

    NASA Astrophysics Data System (ADS)

    Sun, Yan-Qiong; Zhong, Jie-Cen; Liu, Le-Hui; Qiu, Xing-Tai; Chen, Yi-Ping

    2016-11-01

    An organo-bismuth benzoate with phen as auxiliary ligand, [Bi(phen)(C6H5COO)(C6H4COO)] (1) (phen = 1,10-phenanthroline) has been hydrothermally synthesized from bismuth nitrate, 2-mercaptonbenzoic acid with phen as auxiliary ligand and characterized by single-crystal X-ray diffraction, elemental analyses, PXRD, IR spectra, TG analyses, temperature-depended 2D-IR COS (two-dimensional infrared correlation spectroscopy). Interestingly, benzoate anions in 1 came from the desulfuration reaction of 2-mercaptonbenzoic acid under hydrothermal condition. Compound 1 is a discrete organo-bismuth compound with benzoate and phen ligands. The offset face-to-face π-π stacking interactions and C-H⋯O hydrogen bonds link the isolate complex into a 3D supramolecular network. The temperature-depended 2D-IR COS indicates that the stretching vibrations of Cdbnd C/Cdbnd N of aromatic rings and Cdbnd O bonds are sensitive to the temperature change.

  9. 2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

    SciTech Connect

    Ghosh, Ayanjeet E-mail: gai@sas.upenn.edu; Gai, Feng E-mail: gai@sas.upenn.edu; Hochstrasser, Robin M.; Wang, Jun; DeGrado, William F.; Moroz, Yurii S.; Korendovych, Ivan V.; Zanni, Martin

    2014-06-21

    Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs.

  10. Graphene as a substrate to suppress fluorescence in resonance Raman spectroscopy.

    PubMed

    Xie, Liming; Ling, Xi; Fang, Yuan; Zhang, Jin; Liu, Zhongfan

    2009-07-29

    We have measured resonance Raman spectra with greatly suppressed fluorescence (FL) background from rhodamine 6G (R6G) and protoporphyrin IX (PPP) adsorbed on graphene. The FL suppression is estimated to be approximately 10(3) times for R6G. The successful observation of resonance Raman peaks demonstrates that graphene can be used as a substrate to suppress FL in resonance Raman spectroscopy (RRS), which has potential applications in low-concentration detection and RRS study of fluorescent molecules.

  11. Fluorescent probes for shock compression spectroscopy of microstructured materials

    NASA Astrophysics Data System (ADS)

    Christensen, James M.; Banishev, Alexandr A.; Dlott, Dana D.

    2017-01-01

    We are developing fluorescent probes to obtain dynamic two-dimensional pressure maps of shocked microstructured materials. We have fabricated silica nano-or micro-spheres doped with rhodamine 6G dye (R6G) which fluoresce strongly, and which may be dispersed throughout a microstructured sample. Alternatively we can grow thin skin layers of dye-doped silica on the surface of particles. The emissive microspheres were embedded in poly-methyl methacrylate (PMMA) and were excited by a quasi-continuous laser. When the samples were shocked to 3-8.4 GPa using laser-driven flyer plates, the emission redshifted and lost intensity. When encapsulating the dye in silica, the emission became brighter and the intensity-loss response became fast enough to monitor nanosecond shock effects. Preliminary data are reported showing the intensity loss in a shocked microstructured medium, an artificial sand, consisting of dye-coated silica microspheres.

  12. Time-resolved fluorescence spectroscopy for chemical sensors.

    PubMed

    Draxler, S; Lippitsch, M E

    1996-07-20

    A family of sensors is presented with fluorescence decay-time measurements used as the sensing technique. The concept is to take a single fluorophore with a suitably long fluorescence decay time as the basic building block for numerous different sensors. Analyte recognition can be performed by different functional groups that are necessary for selective interaction with the analyte. To achieve this, the principle of excited-state electron transfer is applied with pyrene as the fluorophore. Therefore the same instrumentation based on a small, ambient air-nitrogen laser and solid-state electronics can be used to measure different analytes, for example, oxygen, pH, carbon dioxide, potassium, ammonium, lead, cadmium, zinc, and phosphate.

  13. Time-resolved fluorescence spectroscopy for chemical sensors

    NASA Astrophysics Data System (ADS)

    Draxler, Sonja; Lippitsch, Max E.

    1996-07-01

    A family of sensors is presented with fluorescence decay-time measurements used as the sensing technique. The concept is to take a single fluorophore with a suitably long fluorescence decay time as the basic building block for numerous different sensors. Analyte recognition can be performed by different functional groups that are necessary for selective interaction with the analyte. To achieve this, the principle of excited-state electron transfer is applied with pyrene as the fluorophore. Therefore the same instrumentation based on a small, ambient air-nitrogen laser and solid-state electronics can be used to measure different analytes, for example, oxygen, pH, carbon dioxide, potassium, ammonium, lead, cadmium, zinc, and phosphate.

  14. Investigation of the interaction between five alkaloids and human hemoglobin by fluorescence spectroscopy and molecular modeling.

    PubMed

    He, Wu; Dou, Huanjing; Li, Zhigang; Wang, Xiaogai; Wang, Lvjing; Wang, Ruiyong; Chang, Junbiao

    2014-04-05

    This work studied the interaction of human hemoglobin (HHb) with aminophylline, acefylline, caffeine, theophylline and diprophylline systematically by UV-vis absorption spectroscopy and fluorescence spectroscopy in combination with molecular modeling. Five alkaloids caused the fluorescence quenching of HHb by the formation of alkaloids-HHb complex. The binding constants and thermodynamic parameters were obtained. The hydrophobic and electrostatic interactions were the predominant intermolecular forces to stabilize these complexes. Results of thermodynamic analysis and molecular modeling showed that aminophylline was the strongest quencher and diprophylline was the weakest quencher.

  15. Quantitative frequency-domain fluorescence spectroscopy in tissues and tissue-like media

    NASA Astrophysics Data System (ADS)

    Cerussi, Albert Edward

    1999-09-01

    In the never-ending quest for improved medical technology at lower cost, modern near-infrared optical spectroscopy offers the possibility of inexpensive technology for quantitative and non-invasive diagnoses. Hemoglobin is the dominant chromophore in the 700-900 nm spectral region and as such it allows for the optical assessment of hemoglobin concentration and tissue oxygenation by absorption spectroscopy. However, there are many other important physiologically relevant compounds or physiological states that cannot be effectively sensed via optical methods because of poor optical contrast. In such cases, contrast enhancements are required. Fluorescence spectroscopy is an attractive component of optical tissue spectroscopy. Exogenous fluorophores, as well as some endogenous ones, may furnish the desperately needed sensitivity and specificity that is lacking in near-infrared optical tissue spectroscopy. The main focus of this thesis was to investigate the generation and propagation of fluorescence photons inside tissues and tissue-like media (i.e., scattering dominated media). The standard concepts of fluorescence spectroscopy have been incorporated into a diffusion-based picture that is sometimes referred to as photon migration. The novelty of this work lies in the successful quantitative recovery of fluorescence lifetimes, absolute fluorescence quantum yields, fluorophore concentrations, emission spectra, and both scattering and absorption coefficients at the emission wavelength from a tissue-like medium. All of these parameters are sensitive to the fluorophore local environment and hence are indicators of the tissue's physiological state. One application demonstrating the capabilities of frequency-domain lifetime spectroscopy in tissue-like media is a study of the binding of ethidium bromide to bovine leukocytes in fresh milk. Ethidium bromide is a fluorescent dye that is commonly used to label DNA, and hence visualize chromosomes in cells. The lifetime of

  16. Impurity studies in fusion devices using laser-fluorescence-spectroscopy

    SciTech Connect

    Husinsky, W.R.

    1980-08-01

    Resonance fluorescence excitation of neutral atoms using tunable radiation from dye lasers offers a number of unique advantages for impurity studies in fusion devices. Using this technique, it is possible to perform local, time-resolved measurements of the densities and velocity distributions of metallic impurities in fusion devices without disturbing the plasma. Velocities are measured by monitoring the fluorescence intensity while tuning narrow bandwidth laser radiation through the Doppler - broadened absorbtion spectrum of the transition. The knowledge of the velocity distribution of neutral impurities is particularly useful for the determination of impurity introduction mechanisms. The laser fluorescence technique will be described in terms of its application to metallic impurities in fusion devices and related laboratory experiments. Particular attention will be given to recent results from the ISX-B tokamak using pulsed dye lasers where detection sensitivities for neutral Fe of 10/sup 6/ atoms/cm/sup 3/ with a velocity resolution of 600 m/sec (0.1 eV) have been achieved. Techniques for exciting plasma particles (H,D) will also be discussed.

  17. Noncontact point spectroscopy guided by two-channel fluorescence imaging in a hamster cheek pouch model

    NASA Astrophysics Data System (ADS)

    Yang, Victor X.; Yeow, Jenny; Lilge, Lothar D.; Kost, James; Mang, Thomas S.; Wilson, Brian C.

    1999-07-01

    A system for in vivo, fluorescence image-guided, non-contact point fluorescence spectroscopy is presented. A 442 nm HeCd laser is used as the fluorescence excitation source. An intensified CCD serves as the detector for both imaging and spectroscopy, on which two regions of 300 X 300 pixels were used for green (500 +/- 18 nm) and red (630 +/- 18 nm) imaging channels, and a strip of 600 X 120 pixels are used for emission spectroscopy (450 - 750 nm). At a working distance of 40 mm, the system has a spatial resolution of 0.16 mm and a spectral resolution of 5 nm. System performance is demonstrated in a carcinogenesis model in hamsters, where tumors were induced by painting DMBA in the cheek pouch. Autofluorescence and Photofrin-induced fluorescence measurements were performed every 2 weeks during the 18 weeks of tumor induction. Punch biopsies on selected animals were taken for histological staging. The results show that autofluorescence fluorescence can distinguish dysplasia from normal mucosal tissue model, utilizing the peak red intensity (or the red-to-green intensity ratio). Photofrin-induced fluorescence was superior to autofluorescence for differentiating high grade dysplasia from invasive cancer.

  18. Statistical analysis of excitation-emission matrices for laser-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Maslov, N. A.; Papaeva, E. O.

    2016-07-01

    An algorithm for statistical processing of the set of multicomponent excitation-emission matrices for laser-induced fluorescence spectroscopy is proposed that is based on principal component analysis. It is shown for the first time that the fluorescence emission and excitation spectra of unknown fluorophores in optically thin samples can be calculated. Using the proposed algorithm, it is possible to pass from principal components with alternating signs to positive quantities corresponding to the spectra of real substances. The method is applied to a mixture of three fluorescent dyes, and it is demonstrated that the obtained spectra of principal components well reproduce the spectra of initial dyes.

  19. Comparison of fluorescence spectroscopy and FTIR in differentiation of plant pollens

    NASA Astrophysics Data System (ADS)

    Mularczyk-Oliwa, Monika; Bombalska, Aneta; Kaliszewski, Miron; Włodarski, Maksymilian; Kopczyński, Krzysztof; Kwaśny, Mirosław; Szpakowska, Małgorzata; Trafny, Elżbieta A.

    2012-11-01

    Spectroscopic techniques are under investigation on possibility of differentiation of airborne particles. This paper describes pollen discrimination among others bio-particles in laboratory conditions. Pollen samples were characterized with UV-Vis fluorescence, drift and KBr pellet techniques of infrared spectroscopy. Principal Component Analysis of UV-Vis fluorescence and FTIR spectra revealed that pollens can be distinguished from other bio-materials with use of these methods. Both methods resulted in similar classification capability. Combined FTIR and fluorescence data analysis did not improve the discrimination between pollen allergens and other airborne biological materials.

  20. Automated suppression of sample-related artifacts in Fluorescence Correlation Spectroscopy.

    PubMed

    Ries, Jonas; Bayer, Mathias; Csúcs, Gábor; Dirkx, Ronald; Solimena, Michele; Ewers, Helge; Schwille, Petra

    2010-05-24

    Fluorescence Correlation Spectroscopy (FCS) in cells often suffers from artifacts caused by bright aggregates or vesicles, depletion of fluorophores or bleaching of a fluorescent background. The common practice of manually discarding distorted curves is time consuming and subjective. Here we demonstrate the feasibility of automated FCS data analysis with efficient rejection of corrupted parts of the signal. As test systems we use a solution of fluorescent molecules, contaminated with bright fluorescent beads, as well as cells expressing a fluorescent protein (ICA512-EGFP), which partitions into bright secretory granules. This approach improves the accuracy of FCS measurements in biological samples, extends its applicability to especially challenging systems and greatly simplifies and accelerates the data analysis.

  1. Fluorescence Instrument Response Standards in Two-Photon Time-Resolved Spectroscopy

    PubMed Central

    LUCHOWSKI, RAFAL; SZABELSKI, MARIUSZ; SARKAR, PABAK; APICELLA, ELISA; MIDDE, KRISHNA; RAUT, SANGRAM; BOREJDO, JULIAN; GRYCZYNSKI, ZYGMUNT; GRYCZYNSKI, IGNACY

    2011-01-01

    We studied the fluorescence properties of several potential picosecond lifetime standards suitable for two-photon excitation from a Ti : sapphire femtosecond laser. The fluorescence emission of the selected fluorophores (rose bengal, pyridine 1, and LDS 798) covered the visible to near-infrared wavelength range from 550 to 850 nm. We suggest that these compounds can be used to measure the appropriate instrument response functions needed for accurate deconvolution of fluorescence lifetime data. Lifetime measurements with multiphoton excitation that use scatterers as a reference may fail to properly resolve fluorescence intensity decays. This is because of the different sensitivities of photodetectors in different spectral regions. Also, detectors often lose sensitivity in the near-infrared region. We demonstrate that the proposed references allow a proper reconvolution of measured lifetimes. We believe that picosecond lifetime standards for two-photon excitation will find broad applications in multiphoton spectroscopy and in fluorescence lifetime imaging microscopy (FLIM). PMID:20719056

  2. Determination of dissolved organic matter removal efficiency in wastewater treatment works using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Carstea, Elfrida M.; Bridgeman, John

    2015-04-01

    Fluorescence spectroscopy was used to investigate the removal efficiency of dissolved organic matter (DOM) in several wastewater treatment works, at different processing stages. The correlation between fluorescence values and biochemical oxygen demand (BOD), chemical oxygen demand (COD) and total organic carbon (TOC) has been examined. Fluorescence was measured for unfiltered and filtered (0.45 and 0.20 μm) samples of crude, settled and secondary treated wastewater (activated sludge), and final effluent. Moreover, the potential of using portable fluorimeters has been explored in a laboratory scale activated sludge process. Good correlations were observed for filtered and unfiltered wastewater samples between protein-like fluorescence intensity (excitation 280 nm, emission 350 nm) and BOD (r = 0.78), COD (r = 0.90) and TOC (r = 0.79). BOD displayed a higher correlation at the 0.20 μm filtered samples compared to COD and TOC. Slightly better relation was seen between fluorescence and conventional parameters at the portable fluorimeters compared to laboratory-based instruments. The results indicated that fluorescence spectroscopy, in particular protein-like fluorescence, could be used for continuous, real-time assessment of DOM removal efficiency in wastewater treatment works.

  3. Near-infrared and two-dimensional fluorescence spectroscopy monitoring of monoclonal antibody fermentation media quality: aged media decreases cell growth.

    PubMed

    Hakemeyer, Christian; Strauss, Ulrike; Werz, Silke; Folque, Francisca; Menezes, Jose C

    2013-07-01

    In biomanufacturing processes, the influence of feedstock components on product yield and quality is considerable and often poorly understood. Here we describe the capabilities of near-infrared spectroscopy (NIRS) and two dimensional (2D)-fluorescence spectroscopy in detecting chemical changes over time in two types of culture media (one basal media and one feed media) used in the production of monoclonal antibodies (mAbs) by Chinese hamster ovary (CHO) cells. Both spectroscopies were able to detect compositional changes in basal media over storage period of 12 weeks. NIRS was more effective in detecting changes in feed medium composition. The impact of storage time in process performance was evaluated by using aged media components in mAb cultivations. The study suggests that basal media aging results in a decrease of the integral of viable cells (IVC) (cell growth over time), while product titer is not significantly affected. Feed media appears to be less sensitive to storage and no correlation between the age of the media and cell culture performance was detected. Results obtained provide a basis on which to further improve cell culture raw material quality assessment using vibrational (e.g. NIRS) and optical (e.g. 2D-fluorescence) spectroscopic methods.

  4. Excitation-emission matrices (EEMs) and synchronous fluorescence spectroscopy (SFS) investigations of gastrointestinal tissues

    NASA Astrophysics Data System (ADS)

    Genova, Ts.; Borisova, E.; Zhelyazkova, Al.; Semyachkina-Glushkovskaya, O.; Penkov, N.; Keremedchiev, M.; Vladimirov, B.; Avramov, L.

    2015-01-01

    In this report we will present our recent investigations of the fluorescence properties of lower part gastrointestinal tissues using excitation-emission matrix and synchronous fluorescence spectroscopy measurement modalities. The spectral peculiarities observed will be discussed and the endogenous sources of the fluorescence signal will be addressed. For these fluorescence spectroscopy measurements the FluoroLog 3 system (HORIBA Jobin Yvon, France) was used. It consists of a Xe lamp (300 W, 200-650 nm), a double mono-chromators, and a PMT detector with a work region at 220- 850 nm. Autofluorescence signals were detected in the form of excitation-emission matrices for the samples of normal mucosa, dysphasia and colon carcinoma and specific spectral features for each tissue were found. Autofluorescence signals from the same samples are observed through synchronous fluorescence spectroscopy, which is a novel promising modality for fluorescence spectroscopy measurements of bio-samples. It is one of the most powerful techniques for multicomponent analysis, because of its sensitivity. In the SFS regime, the fluorescence signal is recorded while both excitation λexc and emission wavelengths λem are simultaneously scanned. A constant wavelength interval is maintained between the λexc and λem wavelengths throughout the spectrum. The resulted fluorescence spectrum shows narrower peak widths, in comparison with EEMs, which are easier for identification and minimizes the chance for false determinations or pretermission of specific spectral feature. This modality is also faster, than EEMs, a much smaller number of data points are required.1 In our measurements we use constant wavelength interval Δλ in the region of 10-200 nm. Measurements are carried out in the terms of finding Δλ, which results in a spectrum with most specific spectral features for comparison with spectral characteristics observed in EEMs. Implementing synchronous fluorescence spectroscopy in optical

  5. Picosecond time-resolved fluorescence spectroscopy of phytochrome and stentorin

    NASA Astrophysics Data System (ADS)

    Song, Pill-Soon

    1991-05-01

    Phytochrome is a tetrapyrrole chromoprotein. It serves as a sensitive photosensor for red lightmediated gene expression and other developmental/morphological responses in plants. In this paper photochemical dynamics of the phytochrome molecule have been described in terms of photoisomerization of the tetrapyrrole chromophore in its singlet excited state and subsequent thermal processes in the Pr Pfr phototransformation of phytochrome. Stentorin acts as the photosensor molecule in the ciliate Stentor coeruleus. This unicellular protozoan is most sensitive to red light (610-620 urn). Stentor also senses the direction of light propagation as evidenced by their light-avoiding and negative phototactic swimming behaviors. This aneural photosensory phenomenon is triggered by the photoreceptor stentorin. The possible involvement of a light-induced transient proton release from the photoreceptor as the primary mechanism of light-signal processing has been discussed on the basis of picosecond fluorescence decays and time-resolved fluorescence spectra of stentorin in solution. An initial sensory signal generated by the primary photoprocess of stentorin then triggers subsequent transduction steps that include calcium ion influx from the extracellular medium. Calcium ion influx from the extracellular medium to the cytosol causes the Stentor cell to reverse its ciliary beating and subsequently steer away from the light trap. II.

  6. The roles of carbohydrates, proteins and lipids in the process of aggregation of natural marine organic matter investigated by means of 2D correlation spectroscopy applied to infrared spectra.

    PubMed

    Mecozzi, Mauro; Pietrantonio, Eva; Pietroletti, Marco

    2009-01-01

    In this paper the marine organic matter soluble in an alkaline medium called extractable humic substance (EHS), was extracted from three sediment samples of Tyrrhenian Sea and separated by precipitation at pH 2 in the two fractions of fulvic acids (FAs) and humic acids (HAs). FAs were further fractionated in seven sub-samples of different molecular weight (mw) by means of seven different ultrafiltration membranes operating in the range between mw<1 kDa and mw>100 kDa. Then the qualitative composition of each sample of fractionated FAs and HAs was studied by means of one-dimensional Fourier transform infrared spectroscopy in reflectance mode (FTIR-DRIFT) and by two-dimensional (2D) correlation spectroscopy both in wavelength-wavelength (WW) and in sample-sample (SS) mode. The application of 2D correlation WW spectroscopy allows to elucidate the different roles played by carbohydrates and proteins with respect to some lipid compounds such as fatty acids and ester fatty acids during the process of aggregate formations from mw approximately 1 kDa to higher size aggregates. In addition, 2D correlation WW spectroscopy allows to observe some peculiar interactions between carbohydrates and proteins in the formation of EHS aggregates, interactions which vary from a sample to another sample. The results of 2D correlation SS spectroscopy confirm the general evidences obtained by 2D WW spectroscopy and moreover, they also describe the formation of EHS aggregates as a complex process where evolutionary links and connectivity between aggregates of neighbour molecular size ranges are not evident. Two-dimensional correlation spectroscopy applied to FTIR spectroscopy shows to be a powerful tool for the investigation of the mechanisms involved in EHS aggregation because it supports the acquisition of structural information which sometimes can be hardly obtained by one-dimensional FTIR spectroscopy.

  7. The roles of carbohydrates, proteins and lipids in the process of aggregation of natural marine organic matter investigated by means of 2D correlation spectroscopy applied to infrared spectra

    NASA Astrophysics Data System (ADS)

    Mecozzi, Mauro; Pietrantonio, Eva; Pietroletti, Marco

    2009-01-01

    In this paper the marine organic matter soluble in an alkaline medium called extractable humic substance (EHS), was extracted from three sediment samples of Tyrrhenian Sea and separated by precipitation at pH 2 in the two fractions of fulvic acids (FAs) and humic acids (HAs). FAs were further fractionated in seven sub-samples of different molecular weight (mw) by means of seven different ultrafiltration membranes operating in the range between mw < 1 kDa and mw > 100 kDa. Then the qualitative composition of each sample of fractionated FAs and HAs was studied by means of one-dimensional Fourier transform infrared spectroscopy in reflectance mode (FTIR-DRIFT) and by two-dimensional (2D) correlation spectroscopy both in wavelength-wavelength (WW) and in sample-sample (SS) mode. The application of 2D correlation WW spectroscopy allows to elucidate the different roles played by carbohydrates and proteins with respect to some lipid compounds such as fatty acids and ester fatty acids during the process of aggregate formations from mw ˜1 kDa to higher size aggregates. In addition, 2D correlation WW spectroscopy allows to observe some peculiar interactions between carbohydrates and proteins in the formation of EHS aggregates, interactions which vary from a sample to another sample. The results of 2D correlation SS spectroscopy confirm the general evidences obtained by 2D WW spectroscopy and moreover, they also describe the formation of EHS aggregates as a complex process where evolutionary links and connectivity between aggregates of neighbour molecular size ranges are not evident. Two-dimensional correlation spectroscopy applied to FTIR spectroscopy shows to be a powerful tool for the investigation of the mechanisms involved in EHS aggregation because it supports the acquisition of structural information which sometimes can be hardly obtained by one-dimensional FTIR spectroscopy.

  8. 2D and 3D imaging of the gas phase close to an operating model catalyst by planar laser induced fluorescence

    NASA Astrophysics Data System (ADS)

    Blomberg, Sara; Zhou, Jianfeng; Gustafson, Johan; Zetterberg, Johan; Lundgren, Edvin

    2016-11-01

    In recent years, efforts have been made in catalysis related surface science studies to explore the possibilities to perform experiments at conditions closer to those of a technical catalyst, in particular at increased pressures. Techniques such as high pressure scanning tunneling/atomic force microscopy (HPSTM/AFM), near ambient pressure x-ray photoemission spectroscopy (NAPXPS), surface x-ray diffraction (SXRD) and polarization-modulation infrared reflection absorption spectroscopy (PM-IRAS) at semi-realistic conditions have been used to study the surface structure of model catalysts under reaction conditions, combined with simultaneous mass spectrometry (MS). These studies have provided an increased understanding of the surface dynamics and the structure of the active phase of surfaces and nano particles as a reaction occurs, providing novel information on the structure/activity relationship. However, the surface structure detected during the reaction is sensitive to the composition of the gas phase close to the catalyst surface. Therefore, the catalytic activity of the sample itself will act as a gas-source or gas-sink, and will affect the surface structure, which in turn may complicate the assignment of the active phase. For this reason, we have applied planar laser induced fluorescence (PLIF) to the gas phase in the vicinity of an active model catalysts. Our measurements demonstrate that the gas composition differs significantly close to the catalyst and at the position of the MS, which indeed should have a profound effect on the surface structure. However, PLIF applied to catalytic reactions presents several beneficial properties in addition to investigate the effect of the catalyst on the effective gas composition close to the model catalyst. The high spatial and temporal resolution of PLIF provides a unique tool to visualize the on-set of catalytic reactions and to compare different model catalysts in the same reactive environment. The technique can be

  9. 2D and 3D imaging of the gas phase close to an operating model catalyst by planar laser induced fluorescence.

    PubMed

    Blomberg, Sara; Zhou, Jianfeng; Gustafson, Johan; Zetterberg, Johan; Lundgren, Edvin

    2016-11-16

    In recent years, efforts have been made in catalysis related surface science studies to explore the possibilities to perform experiments at conditions closer to those of a technical catalyst, in particular at increased pressures. Techniques such as high pressure scanning tunneling/atomic force microscopy (HPSTM/AFM), near ambient pressure x-ray photoemission spectroscopy (NAPXPS), surface x-ray diffraction (SXRD) and polarization-modulation infrared reflection absorption spectroscopy (PM-IRAS) at semi-realistic conditions have been used to study the surface structure of model catalysts under reaction conditions, combined with simultaneous mass spectrometry (MS). These studies have provided an increased understanding of the surface dynamics and the structure of the active phase of surfaces and nano particles as a reaction occurs, providing novel information on the structure/activity relationship. However, the surface structure detected during the reaction is sensitive to the composition of the gas phase close to the catalyst surface. Therefore, the catalytic activity of the sample itself will act as a gas-source or gas-sink, and will affect the surface structure, which in turn may complicate the assignment of the active phase. For this reason, we have applied planar laser induced fluorescence (PLIF) to the gas phase in the vicinity of an active model catalysts. Our measurements demonstrate that the gas composition differs significantly close to the catalyst and at the position of the MS, which indeed should have a profound effect on the surface structure. However, PLIF applied to catalytic reactions presents several beneficial properties in addition to investigate the effect of the catalyst on the effective gas composition close to the model catalyst. The high spatial and temporal resolution of PLIF provides a unique tool to visualize the on-set of catalytic reactions and to compare different model catalysts in the same reactive environment. The technique can be

  10. Near-Field Fluorescence Cross-Correlation Spectroscopy on Planar Membranes

    PubMed Central

    2015-01-01

    The organization and dynamics of plasma membrane components at the nanometer scale are essential for biological functions such as transmembrane signaling and endocytosis. Planarized nanoscale apertures in a metallic film are demonstrated as a means of confining the excitation light for multicolor fluorescence spectroscopy to a 55 ± 10 nm beam waist. This technique provides simultaneous two-color, subdiffraction-limited fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy on planar membranes. The fabrication and implementation of this technique are demonstrated for both model membranes and live cells. Membrane-bound proteins were observed to cluster upon the addition of a multivalent cross-linker: On supported lipid bilayers, clusters of cholera toxin subunit B were formed upon cross-linking by an antibody specific for this protein; on living cells, immunoglobulin E bound to its receptor (FcεRI) on the plasma membranes of RBL mast cells was observed to form clusters upon exposure to a trivalent antigen. The formation of membrane clusters was quantified via fluorescence intensity vs time and changes in the temporal auto- and cross-correlations above a single nanoscale aperture. The illumination profile from a single aperture is analyzed experimentally and computationally with a rim-dominated illumination profile, yielding no change in the autocorrelation dwell time with changes in aperture diameter from 60 to 250 nm. This near-field fluorescence cross-correlation methodology provides access to nanoscale details of dynamic membrane interactions and motivates further development of near-field optical methods. PMID:25004429

  11. Improved rovibrational constants for the v7 = 1 state of ethylene-cis-1,2-d2 (cis-C2H2D2) by high-resolution synchrotron FTIR spectroscopy

    NASA Astrophysics Data System (ADS)

    Tan, T. L.; Ng, L. L.; Gabona, M. G.; Aruchunan, G.; Wong, Andy; Appadoo, Dominique R. T.; McNaughton, Don

    2017-01-01

    Using the far-infrared beamline of the Australian Synchrotron, the spectrum of the ν7 band of ethylene-cis-1,2-d2 (cis-C2H2D2) was recorded in the 640-990 cm-1 region at an unapodized resolution of 0.00096 cm-1. A rovibrational analysis of a total of 2823 infrared transitions of the ν7 band was carried out using an asymmetric rotor fitting program based on the Watson's A-reduced Hamiltonian in the Ir representation to derive up to four sextic constants with a rms deviation of 0.00035 cm-1. From the fitting of 2634 ground state combination differences (GSCDs) of cis-C2H2D2 which were derived from the infrared transitions of the ν7 band of this work, and ν10 and ν12 bands of previous studies, together with 22 microwave frequencies, accurate ground state constants of cis-C2H2D2 up to four sextic terms were obtained. The rotational constants (A, B, and C) of the v7 = 1 state of cis-C2H2D2 were found to agree within 0.5% with the calculated values using B3LYP/cc-pVTZ and MP2/cc-pVTZ levels of theory. From this work, the band center of ν7 at 842.209489(20) cm-1 and the rovibrational constants of the v7 = 1 state of cis-C2H2D2 were determined with better accuracy than previously reported.

  12. Capillary Electrophoresis and Fluorescence Excitation-Emission Matrix Spectroscopy for Characterization of Humic Substances

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Capillary electrophoresis (CE) and fluorescence spectroscopy have been used in natural organic matter (NOM) studies. In this study, we characterized five fulvic acids, six humic acids and two unprocessed NOM samples obtained from the International Humic Substances Society (IHSS) using these two ana...

  13. Application of Fluorescence Spectroscopy for Rapid Detection of Pathogens in Food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The potential of fluorescence spectroscopy was investigated for the detection food bone pathogens. E coli, Salmonella and Campylobactor, the most commonly present in food, were selectively identified. Each pathogen, grown in agar plate, was diluted in saline and prepared in different concentrations....

  14. Fluorescence kinetics of Trp-Trp dipeptide and its derivatives in water via ultrafast fluorescence spectroscopy.

    PubMed

    Jia, Menghui; Yi, Hua; Chang, Mengfang; Cao, Xiaodan; Li, Lei; Zhou, Zhongneng; Pan, Haifeng; Chen, Yan; Zhang, Sanjun; Xu, Jianhua

    2015-08-01

    Ultrafast fluorescence dynamics of Tryptophan-Tryptophan (Trp-Trp/Trp2) dipeptide and its derivatives in water have been investigated using a picosecond resolved time correlated single photon counting (TCSPC) apparatus together with a femtosecond resolved upconversion spectrophotofluorometer. The fluorescence decay profiles at multiple wavelengths were fitted by a global analysis technique. Nanosecond fluorescence kinetics of Trp2, N-tert-butyl carbonyl oxygen-N'-aldehyde group-l-tryptophan-l-tryptophan (NBTrp2), l-tryptophan-l-tryptophan methyl ester (Trp2Me), and N-acetyl-l-tryptophan-l-tryptophan methyl ester (NATrp2Me) exhibit multi-exponential decays with the average lifetimes of 1.99, 3.04, 0.72 and 1.22ns, respectively. Due to the intramolecular interaction between two Trp residues, the "water relaxation" lifetime was observed around 4ps, and it is noticed that Trp2 and its derivatives also exhibit a new decay with a lifetime of ∼100ps, while single-Trp fluorescence decay in dipeptides/proteins shows 20-30ps. The intramolecular interaction lifetime constants of Trp2, NBTrp2, Trp2Me and NATrp2Me were then calculated to be 3.64, 0.93, 11.52 and 2.40ns, respectively. Candidate mechanisms (including heterogeneity, solvent relaxation, quasi static self-quenching or ET/PT quenching) have been discussed.

  15. Rapid multiexcitation fluorescence spectroscopy system for in vivo tissue diagnosis

    NASA Astrophysics Data System (ADS)

    Zângaro, Renato Amaro; Silveira, Landulfo, Jr.; Manoharan, Ramasamy; Zonios, George; Itzkan, Irving; Dasari, Ramachandra R.; van Dam, Jacques; Feld, Michael S.

    1996-09-01

    We have designed, fabricated, and tested a compact, transportable, excitation-emission spectrofluorimeter with optical-fiber light delivery and collection for use in rapid analysis of tissues in a clinical setting. This system provides up to eleven different excitation wavelengths, permitting collection of all the corresponding emission spectra in approximately 600 ms. It uses a N2 laser that pumps a sequence of dyes placed in cuvettes on a rotating wheel. A white-light excitation source permits acquisition of the tissue's diffuse reflectance spectrum on each cycle. Return fluorescence and reflected light are dispersed by a small spectrograph and detected by a photodiode-array detector. The system can collect a single-shot spectrum from biological tissue with a signal-to-noise ratio in excess of 50:1.

  16. Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy.

    PubMed

    Gallagher, Sean R

    2017-02-02

    Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing Hoechst 33258, ethidium bromide, and PicoGreen. The range of the assays covers 25 pg/ml to 50 µg/ml. Absorbance at 260 nm has an effective range from 1 to 50 µg/ml; Hoechst 33258 from 0.01 to 15 µg/ml; ethidium bromide from 0.1 to 10 µg/ml; and PicoGreen from 25 to 1000 pg/ml. © 2017 by John Wiley & Sons, Inc.

  17. Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells

    NASA Technical Reports Server (NTRS)

    Malak, H.; Mahtani, H.; Herman, P.; Vecer, J.; Lu, X.; Chang, T. Y.; Richmond, Robert C.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

  18. Intrinsic photosensitizer fluorescence measured using multi-diameter single-fiber spectroscopy in vivo

    NASA Astrophysics Data System (ADS)

    van Leeuwen-van Zaane, Floor; Gamm, Ute A.; van Driel, Pieter B. A. A.; Snoeks, Thomas J.; de Bruijn, Henriette S.; van der Ploeg-van den Heuvel, Angelique; Sterenborg, Henricus J. C. M.; Löwik, Clemens W.; Amelink, Arjen; Robinson, Dominic J.

    2014-01-01

    Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photosensitizers chlorin e6 and Bremachlorin at t=[3,4.5,6,24,48] h incubation time. We observed a tissue-dependent maximum of 35% variation in the total correction factor over the visible wavelength range. Significant differences in spectral shape over time between sensitizers were observed. Although the wavelength position of the fluorescence intensity maximum for ce6 shifted to the red, Bremachlorin showed a blue shift. Furthermore, the Bremachlorin peak appeared to be broader than the ce6 fluorescence peak. Intrinsic fluorescence intensity, which can be related to photosensitizer concentration, was decreasing for all time points but showed significantly more Bremachlorin present compared to ce6 at long incubation times. Results from this study can be used to define an optimal treatment protocol for Bremachlorin-based photodynamic therapy.

  19. Intrinsic photosensitizer fluorescence measured using multi-diameter single-fiber spectroscopy in vivo.

    PubMed

    van Leeuwen-van Zaane, Floor; Gamm, Ute A; van Driel, Pieter B A A; Snoeks, Thomas J; de Bruijn, Henriette S; van der Ploeg-van den Heuvel, Angelique; Sterenborg, Henricus J C M; Löwik, Clemens W; Amelink, Arjen; Robinson, Dominic J

    2014-01-01

    Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photosensitizers chlorin e6 and Bremachlorin at t=[3,4.5,6,24,48]  h incubation time. We observed a tissue-dependent maximum of 35% variation in the total correction factor over the visible wavelength range. Significant differences in spectral shape over time between sensitizers were observed. Although the wavelength position of the fluorescence intensity maximum for ce6 shifted to the red, Bremachlorin showed a blue shift. Furthermore, the Bremachlorin peak appeared to be broader than the ce6 fluorescence peak. Intrinsic fluorescence intensity, which can be related to photosensitizer concentration, was decreasing for all time points but showed significantly more Bremachlorin present compared to ce6 at long incubation times. Results from this study can be used to define an optimal treatment protocol for Bremachlorin-based photodynamic therapy.

  20. Low-dimensional systems investigated by x-ray absorption spectroscopy: a selection of 2D, 1D and 0D cases

    NASA Astrophysics Data System (ADS)

    Mino, Lorenzo; Agostini, Giovanni; Borfecchia, Elisa; Gianolio, Diego; Piovano, Andrea; Gallo, Erik; Lamberti, Carlo

    2013-10-01

    Over the last three decades low-dimensional systems have attracted increasing interest both from the fundamental and technological points of view due to their unique physical and chemical properties. X-ray absorption spectroscopy (XAS) is a powerful tool for the characterization of such kinds of systems, owing to its chemical selectivity and high sensitivity in interatomic distance determination. Moreover, XAS does not require long-range ordering, that is usually absent in low-dimensional systems. Finally, this technique can simultaneously provide information on electronic and local structural properties of the nanomaterials, significantly contributing to clarify the relation between their atomic structure and their peculiar physical properties. This review provides a general introduction to XAS, discussing the basic theory of the technique, the most used detection modes, the related experimental setups and some complementary relevant characterization techniques (diffraction anomalous fine structure, extended energy-loss fine structure, pair distribution function, x-ray emission spectroscopy, high-energy resolution fluorescence detected XAS and x-ray Raman scattering). Subsequently, a selection of significant applications of XAS to two-, one- and zero-dimensional systems will be presented. The selected low-dimensional systems include IV and III-V semiconductor films, quantum wells, quantum wires and quantum dots; carbon-based nanomaterials (epitaxial graphene and carbon nanotubes); metal oxide films, nanowires, nanorods and nanocrystals; metal nanoparticles. Finally, the future perspectives for the application of XAS to nanostructures are discussed.

  1. Fluorescence excitation-emission matrix spectroscopy as a tool for determining quality of sparkling wines.

    PubMed

    Elcoroaristizabal, Saioa; Callejón, Raquel M; Amigo, Jose M; Ocaña-González, Juan A; Morales, M Lourdes; Ubeda, Cristina

    2016-09-01

    Browning in sparkling wines was assessed by the use of excitation-emission fluorescence spectroscopy combined with PARAllel FACtor analysis (PARAFAC). Four different cava sparkling wines were monitored during an accelerated browning process and subsequently storage. Fluorescence changes observed during the accelerated browning process were monitored and compared with other conventional parameters: absorbance at 420nm (A420) and the content of 5-hydroxymethyl-2-furfural (5-HMF). A high similarity of the spectral profiles for all sparkling wines analyzed was observed, being explained by a four component PARAFAC model. A high correlation between the third PARAFAC factor (465/530nm) and the commonly used non-enzymatic browning indicators was observed. The fourth PARAFAC factor (280/380nm) gives us also information about the browning process following a first order kinetic reaction. Hence, excitation-emission fluorescence spectroscopy, together with PARAFAC, provides a faster alternative for browning monitoring to conventional methods, as well as useful key indicators for quality control.

  2. Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution.

    PubMed Central

    Schwille, P; Meyer-Almes, F J; Rigler, R

    1997-01-01

    The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence correlation analysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autocorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence correlation spectroscopy setup and discuss conditions that are favorable for cross-correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-correlation amplitude. Images FIGURE 2 FIGURE 3 PMID:9083691

  3. Use of fluorescence spectroscopy to control ozone dosage in recirculating aquaculture systems.

    PubMed

    Spiliotopoulou, Aikaterini; Martin, Richard; Pedersen, Lars-Flemming; Andersen, Henrik R

    2017-03-15

    The aim of this study was to investigate the potential of fluorescence spectroscopy to be used as an ozone dosage determination tool in recirculating aquaculture systems (RASs), by studying the relationship between fluorescence intensities and dissolved organic matter (DOM) degradation by ozone, in order to optimise ozonation treatment. Water samples from six different Danish facilities (two rearing units from a commercial trout RAS, a commercial eel RAS, a pilot RAS and two marine water aquariums) were treated with different O3 dosages (1.0-20.0 mg/L ozone) in bench-scale experiments, following which fluorescence intensity degradation was eventually determined. Ozonation kinetic experiments showed that RAS water contains fluorescent organic matter, which is easily oxidised upon ozonation in relatively low concentrations (0-5 mg O3/L). Fluorescence spectroscopy has a high level of sensitivity and selectivity in relation to associated fluorophores, and it is able to determine accurately the ozone demand of each system. The findings can potentially be used to design offline or online sensors based on the reduction by ozone of natural fluorescent-dissolved organic matter in RAS. The suggested indirect determination of ozone delivered into water can potentially contribute to a safer and more adequate ozone-based treatment to improve water quality.

  4. Characterising organic matter in recirculating aquaculture systems with fluorescence EEM spectroscopy.

    PubMed

    Hambly, A C; Arvin, E; Pedersen, L-F; Pedersen, P B; Seredyńska-Sobecka, B; Stedmon, C A

    2015-10-15

    The potential of recirculating aquaculture systems (RAS) in the aquaculture industry is increasingly being acknowledged. Along with intensified application, the need to better characterise and understand the accumulated dissolved organic matter (DOM) within these systems increases. Mature RASs, stocked with rainbow trout and operated at steady state at four feed loadings, were analysed by dissolved organic carbon (DOC) analysis and fluorescence excitation-emission matrix (EEM) spectroscopy. The fluorescence dataset was then decomposed by PARAFAC analysis using the drEEM toolbox. This revealed that the fluorescence character of the RAS water could be represented by five components, of which four have previously been identified in fresh water, coastal marine water, wetlands and drinking water. The fluorescence components as well as the DOC showed positive correlations with feed loading, however there was considerable variation between the five fluorescence components with respect to the degree of accumulation with feed loading. The five components were found to originate from three sources: the feed; the influent tap water (groundwater); and processes related to the fish and the water treatment system. This paper details the first application of fluorescence EEM spectroscopy to assess DOM in RAS, and highlights the potential applications of this technique within future RAS management strategies.

  5. Fluorescence spectroscopy and birefringence of molecular changes in maturing rat tail tendon.

    PubMed

    Korol, Renee M; Finlay, Helen M; Josseau, Melanie J; Lucas, Alexandra R; Canham, Peter B

    2007-01-01

    Tissue remodeling during maturation, wound healing, and response to vascular stress involves molecular changes of collagen and elastin in the extracellular matrix (ECM). Two optical techniques are effective for investigating these changes--laser-induced fluorescence (LIF) spectroscopy and polarizing microscopy. LIF spectroscopy integrates the signal from both elastin and collagen cross-linked structure, whereas birefringence is a measure of only collagen. Our purpose is (1) to evaluate the rat tail tendon (RTT) spectroscopy against data from purified extracted protein standards and (2) to correlate the two optical techniques in the study of RTT and skin. Spectra from tissue samples from 27 male rats and from extracted elastin and collagen were obtained using LIF spectroscopy (357 nm). Birefringence was measured on 5-mum histological sections of the same tissue. Morphometric analysis reveals that elastin represents approximately 10% of tendon volume and contributes to RTT fluorescence. RTT maximum fluorescence emission intensity (FEI(max)), which includes collagen and elastin, increases with animal weight (R(2)=0.64). Birefringence, when plotted against weight, increases to a plateau (nonlinear correlation: R(2)=0.90), tendon having greater birefringence than skin. LIF spectroscopy and collagen fiber birefringence are shown to provide complementary measurements of molecular structure (tendon birefringence versus FEI(max) at R(2)=0.60).

  6. Combined fiber probe for fluorescence lifetime and Raman spectroscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Dochow, Sebastian; Ma, Dinglong; Latka, Ines; Bocklitz, Thomas; Hartl, Brad; Bec, Julien; Fatakdawala, Hussain; Wachsmann-Hogiu, Sebastian; Marple, Eric; Urmey, Kirk; Schmitt, Michael; Marcu, Laura; Popp, Jürgen

    2016-03-01

    Raman spectroscopy has been proven to have tremendous potential as biomedical analytical tool for spectroscopic disease diagnostics. The use of fiberoptic coupled Raman spectroscopy systems can enable in-vivo characterization of suspicious lesions. However, Raman spectroscopy has the drawback of rather long acquisition times of several hundreds of milliseconds which makes scanning of larger regions quite challenging. By combining Raman spectroscopy with a fast imaging technique this problem can be alleviate in part. Fluorescence lifetime imaging (FLIm) offers a great potential for such a combination. FLIm can allow for fast tissue area pre-segmentation and location of the points for Raman spectra acquisition. Here, we introduce an optical fiber probe combining FLIm and Raman spectroscopy with an outer diameter of 2 mm. Fluorescence is generated via excitation with a fiber laser at 355 nm. The fluorescence emission is spectrally resolved using a custom-made wavelength-selection module (WSM). The Raman excitation power at 785 nm was set to 50 mW for the in-vivo measurements to prevent sample drying. The lateral probe resolution was determined to be <250 μm for both modalities. This value was taken as step size for several raster scans of different tissue types which were conducted to show the overlap of both modalities under realistic conditions. Finally the probe was used for in vivo raster scans of a rat's brain and subsequently to acquire FLIm guided Raman spectra of several tissues in and around the craniotomy.

  7. Cellulose Structural Polymorphism in Plant Primary Cell Walls Investigated by High-Field 2D Solid-State NMR Spectroscopy and Density Functional Theory Calculations.

    PubMed

    Wang, Tuo; Yang, Hui; Kubicki, James D; Hong, Mei

    2016-06-13

    The native cellulose of bacterial, algal, and animal origins has been well studied structurally using X-ray and neutron diffraction and solid-state NMR spectroscopy, and is known to consist of varying proportions of two allomorphs, Iα and Iβ, which differ in hydrogen bonding, chain packing, and local conformation. In comparison, cellulose structure in plant primary cell walls is much less understood because plant cellulose has lower crystallinity and extensive interactions with matrix polysaccharides. Here we have combined two-dimensional magic-angle-spinning (MAS) solid-state nuclear magnetic resonance (solid-state NMR) spectroscopy at high magnetic fields with density functional theory (DFT) calculations to obtain detailed information about the structural polymorphism and spatial distributions of plant primary-wall cellulose. 2D (13)C-(13)C correlation spectra of uniformly (13)C-labeled cell walls of several model plants resolved seven sets of cellulose chemical shifts. Among these, five sets (denoted a-e) belong to cellulose in the interior of the microfibril while two sets (f and g) can be assigned to surface cellulose. Importantly, most of the interior cellulose (13)C chemical shifts differ significantly from the (13)C chemical shifts of the Iα and Iβ allomorphs, indicating that plant primary-wall cellulose has different conformations, packing, and hydrogen bonding from celluloses of other organisms. 2D (13)C-(13)C correlation experiments with long mixing times and with water polarization transfer revealed the spatial distributions and matrix-polysaccharide interactions of these cellulose structures. Celluloses f and g are well mixed chains on the microfibril surface, celluloses a and b are interior chains that are in molecular contact with the surface chains, while cellulose c resides in the core of the microfibril, outside spin diffusion contact with the surface. Interestingly, cellulose d, whose chemical shifts differ most significantly from those of

  8. Cellulose Structural Polymorphism in Plant Primary Cell Walls Investigated by High-Field 2D Solid-State NMR Spectroscopy and Density Functional Theory Calculations

    PubMed Central

    Wang, Tuo; Yang, Hui; Kubicki, James D.; Hong, Mei

    2017-01-01

    The native cellulose of bacterial, algal, and animal origins has been well studied structurally using X-ray and neutron diffraction and solid-state NMR spectroscopy, and is known to consist of varying proportions of two allomorphs, Iα and Iβ, which differ in hydrogen bonding, chain packing, and local conformation. In comparison, cellulose structure in plant primary cell walls is much less understood because plant cellulose has lower crystallinity and extensive interactions with matrix polysaccharides. Here we have combined two-dimensional magic-angle-spinning (MAS) solid-state nuclear magnetic resonance (solid-state NMR) spectroscopy at high magnetic fields with density functional theory (DFT) calculations to obtain detailed information about the structural polymorphism and spatial distributions of plant primary-wall cellulose. 2D 13C-13C correlation spectra of uniformly 13C-labeled cell walls of several model plants resolved seven sets of cellulose chemical shifts. Among these, five sets (denoted a-e) belong to cellulose in the interior of the microfibril while two sets (f and g) can be assigned to surface cellulose. Importantly, most of the interior cellulose 13C chemical shifts differ significantly from the 13C chemical shifts of the Iα and Iβ allomorphs, indicating that plant primary-wall cellulose has different conformations, packing and hydrogen bonding from celluloses of other organisms. 2D 13C-13C correlation experiments with long mixing times and with water polarization transfer revealed the spatial distributions and matrix-polysaccharide interactions of these cellulose structures. Cellulose f and g are well mixed chains on the microfibril surface, cellulose a and b are interior chains that are in molecular contact with the surface chains, while cellulose c resides in the core of the microfibril, outside spin diffusion contact with the surface. Interestingly, cellulose d, whose chemical shifts differ most significantly from those of bacterial, algal

  9. Parallel β-sheet vibrational couplings revealed by 2D IR spectroscopy of an isotopically labeled macrocycle: quantitative benchmark for the interpretation of amyloid and protein infrared spectra.

    PubMed

    Woys, Ann Marie; Almeida, Aaron M; Wang, Lu; Chiu, Chi-Cheng; McGovern, Michael; de Pablo, Juan J; Skinner, James L; Gellman, Samuel H; Zanni, Martin T

    2012-11-21

    Infrared spectroscopy is playing an important role in the elucidation of amyloid fiber formation, but the coupling models that link spectra to structure are not well tested for parallel β-sheets. Using a synthetic macrocycle that enforces a two stranded parallel β-sheet conformation, we measured the lifetimes and frequency for six combinations of doubly (13)C═(18)O labeled amide I modes using 2D IR spectroscopy. The average vibrational lifetime of the isotope labeled residues was 550 fs. The frequencies of the labels ranged from 1585 to 1595 cm(-1), with the largest frequency shift occurring for in-register amino acids. The 2D IR spectra of the coupled isotope labels were calculated from molecular dynamics simulations of a series of macrocycle structures generated from replica exchange dynamics to fully sample the conformational distribution. The models used to simulate the spectra include through-space coupling, through-bond coupling, and local frequency shifts caused by environment electrostatics and hydrogen bonding. The calculated spectra predict the line widths and frequencies nearly quantitatively. Historically, the characteristic features of β-sheet infrared spectra have been attributed to through-space couplings such as transition dipole coupling. We find that frequency shifts of the local carbonyl groups due to nearest neighbor couplings and environmental factors are more important, while the through-space couplings dictate the spectral intensities. As a result, the characteristic absorption spectra empirically used for decades to assign parallel β-sheet secondary structure arises because of a redistribution of oscillator strength, but the through-space couplings do not themselves dramatically alter the frequency distribution of eigenstates much more than already exists in random coil structures. Moreover, solvent exposed residues have amide I bands with >20 cm(-1) line width. Narrower line widths indicate that the amide I backbone is solvent

  10. Fluorescence spectroscopy incorporating a ratiometric approach for the diagnosis and classification of urothelial carcinoma

    NASA Astrophysics Data System (ADS)

    Anand, Suresh; Cicchi, Riccardo; Crisci, Alfonso; Nesi, Gabriella; Carini, Marco; Pavone, Francesco S.

    2016-02-01

    The current most popular clinical method for the screening of urothelial carcinoma is white light cystoscopy. This method has inherent disadvantages making a strong genesis towards developing more powerful diagnostic techniques. Laser induced intrinsic fluorescence spectroscopy has been studied as an adjunct to current methods for the detection of tumors. This technique allows real time results based on the changes in spectral profile between normal and tumor tissues. We conducted a pilot study based on fluorescence spectroscopy at two wavelengths 378 and 445 nm excitation for the differentiation of urothelial carcinoma. At both the excitation wavelengths, the measured fluorescence signal showed an increased intensity at wavelengths greater than 520 nm. In addition, the emission profile showed modulation at 580 nm which is due to the reabsorption of emitted fluo- rescence due to hemoglobin. Additionally, we developed a tissue characterizing algorithm, based on fluorescence intensity ratios, F510/F600 and F520/F580 at 378 and 445 nm excitation wavelengths respectively. Further, the results were correlated with the pathologists assessment of urothelial carcinoma. This ratiometric classification algorithm yielded 81% sensitivity and 83% specificity at 378 nm and while at 445 nm excitation we achieved a sensitivity and specificity of 85% and 86% for classifying normal and tumor bladder tissues. In this study we have demonstrated the potential of a simple ratiometric algorithm based on fluorescence spectroscopy could be an alternative tool to tissue biopsy. Furthermore, this technique based fiber-based fluorescence spectroscopy could be integrated into an endoscopy system for use in the operating room.

  11. Two-dimensional laser induced fluorescence spectroscopy of van der Waals complexes: fluorobenzene-Ar(n) (n = 1,2).

    PubMed

    Gascooke, Jason R; Alexander, Ula N; Lawrance, Warren D

    2012-04-07

    The technique of two-dimensional laser induced fluorescence (2D-LIF) spectroscopy has been used to observe the van der Waals complexes fluorobenzene-Ar and fluorobenzene-Ar(2) in the region of their S(1)-S(0) electronic origins. The 2D-LIF spectral images reveal a number of features assigned to the van der Waals vibrations in S(0) and S(1). An advantage of 2D-LIF spectroscopy is that the LIF spectrum associated with a particular species may be extracted from an image. This is illustrated for fluorobenzene-Ar. The S(1) van der Waals modes observed in this spectrum are consistent with previous observations using mass resolved resonance enhanced multiphoton ionisation techniques. For S(0), the two bending modes previously observed using a Raman technique were observed along with three new levels. These agree exceptionally well with ab initio calculations. The Fermi resonance between the stretch and bend overtone has been analysed in both the S(0) and S(1) states, revealing that the coupling is stronger in S(0) than in S(1). For fluorobenzene-Ar(2) the 2D-LIF spectral image reveals the S(0) symmetric stretch van der Waals vibration to be 35.0 cm(-1), closely matching the value predicted based on the fluorobenzene-Ar van der Waals stretch frequency. Rotational band contour analysis has been performed on the fluorobenzene-Ar 0(0)(0) transition to yield a set of S(1) rotational constants A' = 0.05871 ± 0.00014 cm(-1), B' = 0.03803 ± 0.00010 cm(-1), and C' = 0.03103 ± 0.00003 cm(-1). The rotational constants imply that in the S(1) 0(0) level the Ar is on average 3.488 Å from the fluorobenzene centre of mass and displaced from it towards the centre of the ring at an angle of ~6° to the normal. The rotational contour for fluorobenzene-Ar(2) was predicted using rotational constants calculated on the basis of the fluorobenzene-Ar geometry and compared with the experimental contour. The comparison is poor which, while due in part to expected saturation effects, suggests

  12. Two-dimensional laser induced fluorescence spectroscopy of van der Waals complexes: Fluorobenzene-Arn (n = 1,2)

    NASA Astrophysics Data System (ADS)

    Gascooke, Jason R.; Alexander, Ula N.; Lawrance, Warren D.

    2012-04-01

    The technique of two-dimensional laser induced fluorescence (2D-LIF) spectroscopy has been used to observe the van der Waals complexes fluorobenzene-Ar and fluorobenzene-Ar2 in the region of their S1-S0 electronic origins. The 2D-LIF spectral images reveal a number of features assigned to the van der Waals vibrations in S0 and S1. An advantage of 2D-LIF spectroscopy is that the LIF spectrum associated with a particular species may be extracted from an image. This is illustrated for fluorobenzene-Ar. The S1 van der Waals modes observed in this spectrum are consistent with previous observations using mass resolved resonance enhanced multiphoton ionisation techniques. For S0, the two bending modes previously observed using a Raman technique were observed along with three new levels. These agree exceptionally well with ab initio calculations. The Fermi resonance between the stretch and bend overtone has been analysed in both the S0 and S1 states, revealing that the coupling is stronger in S0 than in S1. For fluorobenzene-Ar2 the 2D-LIF spectral image reveals the S0 symmetric stretch van der Waals vibration to be 35.0 cm-1, closely matching the value predicted based on the fluorobenzene-Ar van der Waals stretch frequency. Rotational band contour analysis has been performed on the fluorobenzene-Ar overline {0_0^0 } transition to yield a set of S1 rotational constants A' = 0.05871 ± 0.00014 cm-1, B' = 0.03803 ± 0.00010 cm-1, and C' = 0.03103 ± 0.00003 cm-1. The rotational constants imply that in the S1 00 level the Ar is on average 3.488 Å from the fluorobenzene centre of mass and displaced from it towards the centre of the ring at an angle of ˜6° to the normal. The rotational contour for fluorobenzene-Ar2 was predicted using rotational constants calculated on the basis of the fluorobenzene-Ar geometry and compared with the experimental contour. The comparison is poor which, while due in part to expected saturation effects, suggests the presence of another band

  13. A dark-field scanning spectroscopy platform for localized scatter and fluorescence imaging of tissue

    NASA Astrophysics Data System (ADS)

    Krishnaswamy, Venkataramanan; Laughney, Ashley M.; Paulsen, Keith D.; Pogue, Brian W.

    2011-03-01

    Tissue ultra-structure and molecular composition provide native contrast mechanisms for discriminating across pathologically distinct tissue-types. Multi-modality optical probe designs combined with spatially confined sampling techniques have been shown to be sensitive to this type of contrast but their extension to imaging has only been realized recently. A modular scanning spectroscopy platform has been developed to allow imaging localized morphology and molecular contrast measures in breast cancer surgical specimens. A custom designed dark-field telecentric scanning spectroscopy system forms the core of this imaging platform. The system allows imaging localized elastic scatter and fluorescence measures over fields of up to 15 mm x 15 mm at 100 microns resolution in tissue. Results from intralipid and blood phantom measurements demonstrate the ability of the system to quantify localized scatter parameters despite significant changes in local absorption. A co-registered fluorescence spectroscopy mode is also demonstrated in a protophorphyrin-IX phantom.

  14. Fluorescent core-shell star polymers based bioassays for ultrasensitive DNA detection by surface plasmon fluorescence spectroscopy.

    PubMed

    Feng, Chuan Liang; Yin, Meizhen; Zhang, Di; Zhu, Shenmin; Caminade, Anne Marie; Majoral, Jean Pierre; Müllen, Klaus

    2011-04-19

    Multilayers containing a perylene diimide labelled star polymers (FSP) donor adjacent to phosphorus dendrimer layer on a silver substrate were constructed by layer by layer (LBL) approach. Using Surface Plasmon Enhanced Fluorescence Spectroscopy (SPFS) technique, a time-resolved ultrasensitive and selective detection of DNA targets relying on enhanced optical fields associated with energy transfer (ET) were achieved under the excitation at 543 nm. The detection limit is about 8 orders of magnitude better than the achieved one under the excitation at 632 nm, which is ascribed to no energy transfer from the donor to the acceptor under the excitation at 632 nm, resulting in much weak detection signal in turn.

  15. Feasibility of Raman spectroscopy in vitro after 5-ALA-based fluorescence diagnosis in the bladder

    NASA Astrophysics Data System (ADS)

    Grimbergen, M. C. M.; van Swol, C. F. P.; van Moorselaar, R. J. A.; Mahadevan-Jansen, A.,; Stone, N.

    2006-02-01

    Photodynamic diagnosis (PDD) has become popular in bladder cancer detection. Several studies have however shown an increased false positive biopsies rate under PDD guidance compared to conventional cystoscopy. Raman spectroscopy is an optical technique that utilizes molecular specific, inelastic scattering of light photons to interrogate biological tissues, which can successfully differentiate epithelial neoplasia from normal tissue and inflammations in vitro. This investigation was performed to show the feasibility of NIR Raman spectroscopy in vitro on biopsies obtained under guidance of 5-ALA induced PPIX fluorescence imaging. Raman spectra of a PPIX solution was measured to obtain a characteristic signature for the photosensitzer without contributions from tissue constituents. Biopsies were obtained from patients with known bladder cancer instilled with 50ml, 5mg 5-ALA two hours prior to trans-urethral resection of tumor (TURT). Additional biopsies were obtained at a fluorescent and non-fluorescent area, snap-frozen in liquid nitrogen and stored at -80 °C. Each biopsy was thawed before measurements (10sec integration time) with a confocal Raman system (Renishaw Gloucestershire, UK). The 830 nm excitation (300mW) source is focused on the tissue by a 20X ultra-long-working-distance objective. Differences in fluorescence background between the two groups were removed by means of a special developed fluorescence subtraction algorithm. Raman spectra from ALA biopsies showed different fluorescence background which can be effectively removed by a fluorescence subtraction algorithm. This investigation shows that the interaction of the ALA induced PPIX with Raman spectroscopy in bladder samples. Combination of these techniques in-vivo may lead to a viable method of optical biopsies in bladder cancer detection.

  16. Quantification of transition dipole strengths using 1D and 2D spectroscopy for the identification of molecular structures via exciton delocalization: Application to α-helices

    PubMed Central

    Grechko, Maksim; Zanni, Martin T.

    2012-01-01

    Vibrational and electronic transition dipole strengths are often good probes of molecular structures, especially in excitonically coupled systems of chromophores. One cannot determine transition dipole strengths using linear spectroscopy unless the concentration is known, which in many cases it is not. In this paper, we report a simple method for measuring transition dipole moments from linear absorption and 2D IR spectra that does not require knowledge of concentrations. Our method is tested on several model compounds and applied to the amide I′ band of a polypeptide in its random coil and α-helical conformation as modulated by the solution temperature. It is often difficult to confidently assign polypeptide and protein secondary structures to random coil or α-helix by linear spectroscopy alone, because they absorb in the same frequency range. We find that the transition dipole strength of the random coil state is 0.12 ± 0.013 D2, which is similar to a single peptide unit, indicating that the vibrational mode of random coil is localized on a single peptide unit. In an α-helix, the lower bound of transition dipole strength is 0.26 ± 0.03 D2. When taking into account the angle of the amide I′ transition dipole vector with respect to the helix axis, our measurements indicate that the amide I′ vibrational mode is delocalized across a minimum of 3.5 residues in an α-helix. Thus, one can confidently assign secondary structure based on exciton delocalization through its effect on the transition dipole strength. Our method will be especially useful for kinetically evolving systems, systems with overlapping molecular conformations, and other situations in which concentrations are difficult to determine. PMID:23163364

  17. Laser-induced fluorescence-cued, laser-induced breakdown spectroscopy biological-agent detection

    SciTech Connect

    Hybl, John D.; Tysk, Shane M.; Berry, Shaun R.; Jordan, Michael P

    2006-12-01

    Methods for accurately characterizing aerosols are required for detecting biological warfare agents. Currently, fluorescence-based biological agent sensors provide adequate detection sensitivity but suffer from high false-alarm rates. Combining single-particle fluorescence analysis with laser-induced breakdown spectroscopy (LIBS) provides additional discrimination and potentially reduces false-alarm rates. A transportable UV laser-induced fluorescence-cued LIBS test bed has been developed and used to evaluate the utility of LIBS for biological-agent detection. Analysis of these data indicates that LIBS adds discrimination capability to fluorescence-based biological-agent detectors.However, the data also show that LIBS signatures of biological agent simulants are affected by washing. This may limit the specificity of LIBS and narrow the scope of its applicability in biological-agent detection.

  18. Water in Biomolecular Fluorescence Spectroscopy and Imaging: Side Effects and Remedies.

    PubMed

    Fürstenberg, Alexandre

    2017-02-22

    Historically, many of the classical organic fluorescent dyes were developed as laser dyes and characterized and optimized in organic solvents. Since then, fluorescence has, however, found a vast range of applications in the life sciences in which the fluorophores are usually surrounded by water and not by organic solvents. The omnipresence of water in biomolecular fluorescence spectroscopy and imaging leads to some unwanted but nonetheless unavoidable consequences on the photophysical properties of the dyes, which may impact the quality and complicate quantitative interpretation of the experiments. This paper discusses and illustrates with examples two such water-induced phenomena, namely chromophore aggregation in water and fluorescence quenching by water, as well as some ways to overcome them.

  19. Dual-color fluorescence cross-correlation spectroscopy on a planar optofluidic chip.

    PubMed

    Chen, A; Eberle, M M; Lunt, E J; Liu, S; Leake, K; Rudenko, M I; Hawkins, A R; Schmidt, H

    2011-04-21

    Fluorescence cross-correlation spectroscopy (FCCS) is a highly sensitive fluorescence technique with distinct advantages in many bioanalytical applications involving interaction and binding of multiple components. Due to the use of multiple beams, bulk optical FCCS setups require delicate and complex alignment procedures. We demonstrate the first implementation of dual-color FCCS on a planar, integrated optofluidic chip based on liquid-core waveguides that can guide liquid and light simultaneously. In this configuration, the excitation beams are delivered in predefined locations and automatically aligned within the excitation waveguides. We implement two canonical applications of FCCS in the optofluidic lab-on-chip environment: particle colocalization and binding/dissociation dynamics. Colocalization is demonstrated in the detection and discrimination of single-color and double-color fluorescently labeled nanobeads. FCCS in combination with fluorescence resonance energy transfer (FRET) is used to detect the denaturation process of double-stranded DNA at nanomolar concentration.

  20. Rapid screening test for porphyria diagnosis using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Lang, A.; Stepp, H.; Homann, C.; Hennig, G.; Brittenham, G. M.; Vogeser, M.

    2015-07-01

    Porphyrias are rare genetic metabolic disorders, which result from deficiencies of enzymes in the heme biosynthesis pathway. Depending on the enzyme defect, different types of porphyrins and heme precursors accumulate for the different porphyria diseases in erythrocytes, liver, blood plasma, urine and stool. Patients with acute hepatic porphyrias can suffer from acute neuropathic attacks, which can lead to death when undiagnosed, but show only unspecific clinical symptoms such as abdominal pain. Therefore, in addition to chromatographic methods, a rapid screening test is required to allow for immediate identification and treatment of these patients. In this study, fluorescence spectroscopic measurements were conducted on blood plasma and phantom material, mimicking the composition of blood plasma of porphyria patients. Hydrochloric acid was used to differentiate the occurring porphyrins (uroporphyrin-III and coproporphyrin-III) spectroscopically despite their initially overlapping excitation spectra. Plasma phantom mixtures were measured using dual wavelength excitation and the corresponding concentrations of uroporphyrin-III and coproporphyrin-III were determined. Additionally, three plasma samples of porphyria patients were examined and traces of coproporphyrin-III and uroporphyrin-III were identified. This study may therefore help to establish a rapid screening test method with spectroscopic differentiation of the occurring porphyrins, which consequently allows for the distinction of different porphyrias. This may be a valuable tool for clinical porphyria diagnosis and rapid or immediate treatment.

  1. Fluorescence and absorption spectroscopy of the weakly fluorescent chlorophyll a in cytochrome b6f of Synechocystis PCC6803.

    PubMed Central

    Peterman, E J; Wenk, S O; Pullerits, T; Pâlsson, L O; van Grondelle, R; Dekker, J P; Rögner, M; van Amerongen, H

    1998-01-01

    A spectroscopic characterization of the chlorophyll a (Chl) molecule in the monomeric cytochrome b6f complex (Cytb6f) isolated from the cyanobacterium Synechocystis PCC6803 is presented. The fluorescence lifetime and quantum yield have been determined, and it is shown that Chl in Cytb6f has an excited-state lifetime that is 20 times smaller than that of Chl in methanol. This shortening of the Chl excited state lifetime is not caused by an increased rate of intersystem crossing. Most probably it is due to quenching by a nearby amino acid. It is suggested that this quenching is a mechanism for preventing the formation of Chl triplets, which can lead to the formation of harmful singlet oxygen. Using site-selected fluorescence spectroscopy, detailed information on vibrational frequencies in both the ground and Qy excited states has been obtained. The vibrational frequencies indicate that the Chl molecule has one axial ligand bound to its central magnesium and accepts a hydrogen bond to its 13(1)-keto carbonyl. The results show that the Chl binds to a well-defined pocket of the protein and experiences several close contacts with nearby amino acids. From the site-selected fluorescence spectra, it is further concluded that the electron-phonon coupling is moderately strong. Simulations of both the site-selected fluorescence spectra and the temperature dependence of absorption and fluorescence spectra are presented. These simulations indicate that the Huang-Rhys factor characterizing the electron-phonon coupling strength is between 0.6 and 0.9. The width of the Gaussian inhomogeneous distribution function is 210 +/- 10 cm-1. PMID:9649396

  2. Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics.

    PubMed

    Kondo, Tadashi; Hirohashi, Setsuo

    2006-01-01

    Proteome data combined with histopathological information provides important, novel clues for understanding cancer biology and reveals candidates for tumor markers and therapeutic targets. We have established an application of a highly sensitive fluorescent dye (CyDye DIGE Fluor saturation dye), developed for two-dimensional difference gel electrophoresis (2D-DIGE), to the labeling of proteins extracted from laser microdissected tissues. The use of the dye dramatically decreases the protein amount and, in turn, the number of cells required for 2D-DIGE; the cells obtained from a 1 mm2 area of an 8-12 microm thick tissue section generate up to 5,000 protein spots in a large-format 2D gel. This protocol allows the execution of large-scale proteomics in a more efficient, accurate and reproducible way. The protocol can be used to examine a single sample in 5 d or to examine hundreds of samples in large-scale proteomics.

  3. Real time optical Biopsy: Time-resolved Fluorescence Spectroscopy instrumentation and validation

    PubMed Central

    Kittle, David S.; Vasefi, Fartash; Patil, Chirag G.; Mamelak, Adam; Black, Keith L.; Butte, Pramod V.

    2016-01-01

    The Time-resolved fluorescence spectroscopy (TR-FS) has the potential to differentiate tumor and normal tissue in real time during surgical excision. In this manuscript, we describe the design of a novel TR-FS device, along with preliminary data on detection accuracy for fluorophores in a mixture. The instrument is capable of near real-time fluorescence lifetime acquisition in multiple spectral bands and analysis. It is also able to recover fluorescence lifetime with sub-20ps accuracy as validated with individual organic fluorescence dyes and dye mixtures yielding lifetime values for standard fluorescence dyes that closely match with published data. We also show that TR-FS is able to quantify the relative concentration of fluorescence dyes in a mixture by the unmixing of lifetime decays. We show that the TR-FS prototype is able to identify in near-real time the concentrations of dyes in a complex mixture based on previously trained data. As a result, we demonstrate that in complex mixtures of fluorophores, the relative concentration information is encoded in the fluorescence lifetime across multiple spectral bands. We show for the first time the temporal and spectral measurements of a mixture of fluorochromes and the ability to differentiate relative concentrations of each fluorochrome mixture in real time. PMID:27929039

  4. Real time optical Biopsy: Time-resolved Fluorescence Spectroscopy instrumentation and validation.

    PubMed

    Kittle, David S; Vasefi, Fartash; Patil, Chirag G; Mamelak, Adam; Black, Keith L; Butte, Pramod V

    2016-12-08

    The Time-resolved fluorescence spectroscopy (TR-FS) has the potential to differentiate tumor and normal tissue in real time during surgical excision. In this manuscript, we describe the design of a novel TR-FS device, along with preliminary data on detection accuracy for fluorophores in a mixture. The instrument is capable of near real-time fluorescence lifetime acquisition in multiple spectral bands and analysis. It is also able to recover fluorescence lifetime with sub-20ps accuracy as validated with individual organic fluorescence dyes and dye mixtures yielding lifetime values for standard fluorescence dyes that closely match with published data. We also show that TR-FS is able to quantify the relative concentration of fluorescence dyes in a mixture by the unmixing of lifetime decays. We show that the TR-FS prototype is able to identify in near-real time the concentrations of dyes in a complex mixture based on previously trained data. As a result, we demonstrate that in complex mixtures of fluorophores, the relative concentration information is encoded in the fluorescence lifetime across multiple spectral bands. We show for the first time the temporal and spectral measurements of a mixture of fluorochromes and the ability to differentiate relative concentrations of each fluorochrome mixture in real time.

  5. Evaluating Activated Carbon Adsorption of Dissolved Organic Matter and Micropollutants Using Fluorescence Spectroscopy.

    PubMed

    Shimabuku, Kyle K; Kennedy, Anthony M; Mulhern, Riley E; Summers, R Scott

    2017-02-14

    Dissolved organic matter (DOM) negatively impacts granular activated carbon (GAC) adsorption of micropollutants and is a disinfection byproduct precursor. DOM from surface waters, wastewater effluent, and 1 kDa size fractions were adsorbed by GAC and characterized using fluorescence spectroscopy, UV-absorption, and size exclusion chromatography (SEC). Fluorescing DOM was preferentially adsorbed relative to UV-absorbing DOM. Humic-like fluorescence (peaks A and C) was selectively adsorbed relative to polyphenol-like fluorescence (peaks T and B) potentially due to size exclusion effects. In the surface waters and size fractions, peak C was preferentially removed relative to peak A, whereas the reverse was found in wastewater effluent, indicating that humic-like fluorescence is associated with different compounds depending on DOM source. Based on specific UV-absorption (SUVA), aromatic DOM was preferentially adsorbed. The fluorescence index (FI), if interpreted as an indicator of aromaticity, indicated the opposite but exhibited a strong relationship with average molecular weight, suggesting that FI might be a better indicator of DOM size than aromaticity. The influence of DOM intermolecular interactions on adsorption were minimal based on SEC analysis. Fluorescence parameters captured the impact of DOM size on the fouling of 2-methylisoborneol and warfarin adsorption and correlated with direct competition and pore blockage indicators.

  6. Assembly and characterization of a fluorescence lifetime spectroscopy system for skin lesions diagnostic

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Texiera Rosa, Ramon Gabriel; Pratavieira, Sebastião.; D´Almeida, Camila de Paula; Kurachi, Cristina

    2015-06-01

    The fluorescence spectra and fluorescence lifetime analysis in biological tissues has been presented as a technique of a great potential for tissue characterization for diagnostic purposes. The objective of this study is to assemble and characterize a fluorescence lifetime spectroscopy system for diagnostic of clinically similar skin lesions in vivo. The fluorescence lifetime measurements were performed using the Time Correlated Single Photon Counting (Becker & Hickl, Berlin, Germany) technique. Two lasers, one emitting at 378 nm and another at 445 nm, are used for excitation with 20, 50 and 80 MHz repetition rate. A bifurcated optical fiber probe conducts the excitation light to the sample, the collected light is transmitted through bandpass filters and delivered to a hybrid photomultiplier tube detector. The fluorescence spectra were obtained by using a portable spectrometer (Ocean Optics USB-2000-FLG) with the same excitation sources. An instrument response function of about 300 ps was obtained and the spectrum and fluorescence lifetime of a standard fluorescent molecule (Rhodamine 6G) was measured for the calibration of the system ((4.1 +/- 0.3) ns). The assembled system was considered robust, well calibrated and will be used for clinical measurements of skin lesions.

  7. Real time optical Biopsy: Time-resolved Fluorescence Spectroscopy instrumentation and validation

    NASA Astrophysics Data System (ADS)

    Kittle, David S.; Vasefi, Fartash; Patil, Chirag G.; Mamelak, Adam; Black, Keith L.; Butte, Pramod V.

    2016-12-01

    The Time-resolved fluorescence spectroscopy (TR-FS) has the potential to differentiate tumor and normal tissue in real time during surgical excision. In this manuscript, we describe the design of a novel TR-FS device, along with preliminary data on detection accuracy for fluorophores in a mixture. The instrument is capable of near real-time fluorescence lifetime acquisition in multiple spectral bands and analysis. It is also able to recover fluorescence lifetime with sub-20ps accuracy as validated with individual organic fluorescence dyes and dye mixtures yielding lifetime values for standard fluorescence dyes that closely match with published data. We also show that TR-FS is able to quantify the relative concentration of fluorescence dyes in a mixture by the unmixing of lifetime decays. We show that the TR-FS prototype is able to identify in near-real time the concentrations of dyes in a complex mixture based on previously trained data. As a result, we demonstrate that in complex mixtures of fluorophores, the relative concentration information is encoded in the fluorescence lifetime across multiple spectral bands. We show for the first time the temporal and spectral measurements of a mixture of fluorochromes and the ability to differentiate relative concentrations of each fluorochrome mixture in real time.

  8. Method for rapid multidiameter single-fiber reflectance and fluorescence spectroscopy through a fiber bundle.

    PubMed

    Hoy, Christopher L; Gamm, Ute A; Sterenborg, Henricus J C M; Robinson, Dominic J; Amelink, Arjen

    2013-10-01

    We have recently demonstrated a means for quantifying the absorption and scattering properties of biological tissue through multidiameter single-fiber reflectance (MDSFR) spectroscopy. These measurements can be used to correct single-fiber fluorescence (SFF) spectra for the influence of optical properties, enabling quantification of intrinsic fluorescence. In our previous work, we have used a series of pinholes to show that selective illumination and light collection using a coherent fiber bundle can simulate a single solid-core optical fiber with variable diameter for the purposes of MDSFR spectroscopy. Here, we describe the construction and validation of a clinical MDSFR/SFF spectroscopy system that avoids the limitations encountered with pinholes and free-space optics. During one measurement, the new system acquires reflectance spectra at the effective diameters of 200, 600, and 1000 μm, and a fluorescence spectrum at an effective diameter of 1000 μm. From these spectra, we measure the absolute absorption coefficient, μ(a), reduced scattering coefficient, μ'(s'), phase function parameter, γ, and intrinsic fluorescence, Qμ(a,x)(f), across the measured spectrum. We validate the system using Intralipid- and polystyrene sphere-based scattering phantoms, with and without the addition of the absorber Evans Blue. Finally, we demonstrate the combined MDSFR/SFF of phantoms with varying concentrations of Intralipid and fluorescein, wherein the scattering properties are measured by MDSFR and used to correct the SFF spectrum for accurate quantification of Qμ(a,x)(f).

  9. Method for rapid multidiameter single-fiber reflectance and fluorescence spectroscopy through a fiber bundle

    NASA Astrophysics Data System (ADS)

    Amelink, A.; Hoy, C. L.; Gamm, U. A.; Sterenborg, H. J. C. M.; Robinson, D. J.

    2014-03-01

    We have recently demonstrated a means for quantifying the absorption and scattering properties of biological tissue through multidiameter single-fiber reflectance (MDSFR) spectroscopy. These measurements can be used to correct single-fiber fluorescence (SFF) spectra for the influence of optical properties, enabling quantification of intrinsic fluorescence. In our previous work, we have used a series of pinholes to show that selective illumination and light collection using a coherent fiber bundle can simulate a single solid-core optical fiber with variable diameter for the purposes of MDSFR spectroscopy. Here, we describe the construction and validation of a clinical MDSFR/SFF spectroscopy system that avoids the limitations encountered with pinholes and free-space optics. During one measurement, the new system acquires reflectance spectra at the effective diameters of 200, 600, and 1000 μm, and a fluorescence spectrum at an effective diameter of 1000 μm. From these spectra, we measure the absolute absorption coefficient, μa, reduced scattering coefficient, μ's, phase function parameter, γ, and intrinsic fluorescence, Qμfa, across the measured spectrum. We validate the system using Intralipid- and polystyrene sphere-based scattering phantoms, with and without the addition of the absorber Evans Blue. Finally, we demonstrate the combined MDSFR/SFF of phantoms with varying concentrations of Intralipid and fluorescein, wherein the scattering properties are measured by MDSFR and used to correct the SFF spectrum for accurate quantification of Qμfa.

  10. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds.

    PubMed

    Holt, David; Parthasarathy, Ashwin B; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16 . Ten animals showed no residual tumor cells in the wound bed (mean SBR<2 , P<0.001 ). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15 , and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

  11. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

    NASA Astrophysics Data System (ADS)

    Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ˜16. Ten animals showed no residual tumor cells in the wound bed (mean SBR<2, P<0.001). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

  12. Cy3 in AOT reverse micelles II. Probing intermicellar interactions using fluorescence correlation spectroscopy.

    PubMed

    McPhee, Jeffrey T; Scott, Eric; Levinger, Nancy E; Van Orden, Alan

    2011-08-11

    Cyanine-3 (Cy3) fluorescent dye molecules confined in sodium di-2-ethylhexyl sulfosuccinate (AOT) reverse micelles were examined using dynamic light scattering and fluorescence correlation spectroscopy to probe the kinetics of Cy3 dye and reverse micelle aggregation. This study explored a range of reverse micelle sizes, defined as w(0) = [H(2)O]/[AOT], in which the occupation number ranged from one Cy3 molecule per ∼10(5) to ∼10(6) reverse micelles. These measurements reveal that in the smallest reverse micelle, w(0) = 1, the Cy3 molecules aggregate to form H-aggregate dimers, and the Cy3 dimerization is accompanied by the formation of a transient dimer between reverse micelles. Transient reverse micelle dimer particles are only observed in the small fraction of Cy3-labeled reverse micelles probed by fluorescence correlation spectroscopy and are not observed in the bulk solution probed by dynamic light scattering. Furthermore, fluorescence correlation spectroscopy makes it possible to probe the size and shape of these dimers, revealing prolate ellipsoid-shaped particles with twice the volume and surface area of a single reverse micelle.

  13. Optical fluorescence spectroscopy to detect hepatic necrosis after normothermic ischemia: animal model

    NASA Astrophysics Data System (ADS)

    Romano, Renan A.; Vollet-Filho, Jose D.; Pratavieira, Sebastião.; Fernandez, Jorge L.; Kurachi, Cristina; Bagnato, Vanderlei S.; Castro-e-Silva, Orlando; Sankarankutty, Ajith K.

    2015-06-01

    Liver transplantation is a well-established treatment for liver failure. However, the success of the transplantation procedure depends on liver graft conditions. The tissue function evaluation during the several transplantation stages is relevant, in particular during the organ harvesting, when a decision is made concerning the viability of the graft. Optical fluorescence spectroscopy is a good option because it is a noninvasive and fast technique. A partial normothermic hepatic ischemia was performed in rat livers, with a vascular occlusion of both median and left lateral lobes, allowing circulation only for the right lateral lobe and the caudate lobe. Fluorescence spectra under excitation at 532 nm (doubled frequency Nd:YAG laser) were collected using a portable spectrometer (USB2000, Ocean Optics, USA). The fluorescence emission was collected before vascular occlusion, after ischemia, and 24 hours after reperfusion. A morphometric histology analysis was performed as the gold standard evaluation - liver samples were analyzed, and the percentage of necrotic tissue was obtained. The results showed that changes in the fluorescence emission after ischemia can be correlated with the amount of necrosis evaluated by a morphometric analysis, the Pearson correlation coefficient of the generated model was 0.90 and the root mean square error was around 20%. In this context, the laser-induced fluorescence spectroscopy technique after normothermic ischemia showed to be a fast and efficient method to differentiate ischemic injury from viable tissues.

  14. Assessing the photoaging process at sun exposed and non-exposed skin using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Kurachi, Cristina

    2016-03-01

    Photoaging is the skin premature aging due to exposure to ultraviolet light, which damage the collagen, elastin and can induce alterations on the skin cells DNA, and, then, it may evolve to precancerous lesions, which are widely investigated by fluorescence spectroscopy and lifetime. The fluorescence spectra and fluorescence lifetime analysis has been presented as a technique of great potential for biological tissue characterization at optical diagnostics. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and may contribute to a non-invasive clinical investigation of injuries such as skin lesions. These lesions and the possible areas where they may develop can be interrogated using fluorescence lifetime spectroscopy taking into account the variability of skin phototypes and the changes related to melanin, collagen and elastin, endogenous fluorophores which have emissions that spectrally overlap to the NADH and FAD emission. The objective of this study is to assess the variation on fluorescence lifetimes of normal skin at sun exposed and non-exposed areas and associate this variation to the photoaging process.

  15. Towards in situ fluorescence spectroscopy and microscopy investigations of asphaltene precipitation kinetics.

    PubMed

    Franco, Juliana C; Gonçalves, Grasiele; Souza, Monique S; Rosa, Samantha B C; Thiegue, Larissa M; Atvars, Teresa D Z; Rosa, Paulo T V; Nome, René A

    2013-12-16

    We perform a spectroscopic analysis of asphaltene in solution and in crude oil with the goal of designing an optical probe of asphaltene precipitation inside high-pressure cells. Quantitative analysis of steady-state spectroscopic data is employed to identify fluorescence and Raman contributions to the observed signals. Time-resolved fluorescence spectroscopy indicates that fluorescence lifetime can be used as a spectroscopic probe of asphaltene in crude oil. Quantitative confocal laser-scanning microscopy studies of asphaltene in n-heptane are used to calculate particle-size distributions as a function of time, both at the sample surface and asphaltene interior. The resulting precipitation kinetics is well described by stochastic numerical simulations of diffusion-limited aggregation. Based on these results, we present the design and construction of an apparatus to optically probe the in situ precipitation of asphaltene suitable for studies inside high pressure cells. Design considerations include the use of a spatial light modulator for aberration correction in microscopy measurements, together with the design of epi-fluorescence spectrometer, both fiber-based and for remote sensing fluorescence spectroscopy.

  16. Fluorescence suppression using wavelength modulated Raman spectroscopy in fiber-probe-based tissue analysis.

    PubMed

    Praveen, Bavishna B; Ashok, Praveen C; Mazilu, Michael; Riches, Andrew; Herrington, Simon; Dholakia, Kishan

    2012-07-01

    In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (∼30  s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.

  17. Applicability of Fluorescence and Absorbance Spectroscopy to Estimate Organic Pollution in Rivers.

    PubMed

    Knapik, Heloise Garcia; Fernandes, Cristovão Vicente Scapulatempo; de Azevedo, Júlio Cesar Rodrigues; do Amaral Porto, Monica Ferreira

    2014-12-01

    This article explores the applicability of fluorescence and absorbance spectroscopy for estimating organic pollution in polluted rivers. The relationship between absorbance, fluorescence intensity, dissolved organic carbon, biochemical oxygen demand (BOD), chemical oxygen demand (COD), and other water quality parameters were used to characterize and identify the origin and the spatial variability of the organic pollution in a highly polluted watershed. Analyses were performed for the Iguassu River, located in southern Brazil, with area about 2,700 km(2) and ∼3 million inhabitants. Samples were collect at six monitoring sites covering 107 km of the main river. BOD, COD, nitrogen, and phosphorus concentration indicates a high input of sewage to the river. Specific absorbance at 254 and 285 nm (SUVA254 and A285/COD) did not show significant variation between sites monitored, indicating the presence of both dissolved compounds found in domestic effluents and humic and fulvic compounds derived from allochthonous organic matter. Correlations between BOD and tryptophan-like fluorescence peak (peak T2, r=0.7560, and peak T1, r=0.6949) and tyrosine-like fluorescence peak (peak B, r=0.7321) indicated the presence of labile organic matter and thus confirmed the presence of sewage in the river. Results showed that fluorescence and absorbance spectroscopy provide useful information on pollution in rivers from critical watersheds and together are a robust method that is simpler and more rapid than traditional methods employed by regulatory agencies.

  18. Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry.

    PubMed

    Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

    2016-01-15

    Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu(2+) with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15K in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu(2+) ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu(2+) ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu(2+) ions are discussed.

  19. Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry

    NASA Astrophysics Data System (ADS)

    Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

    2016-01-01

    Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu2 + with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15 K in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu2 + ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu2 + ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu2 + ions are discussed.

  20. Determining Individual Mineral Contributions To U(VI) Adsorption In A Contaminated Aquifer Sediment: A Fluorescence Spectroscopy Study

    SciTech Connect

    Wang, Zheming; Zachara, John M.; Boily, Jean F.; Xia, Yuanxian; Resch, Charles T.; Moore, Dean A.; Liu, Chongxuan

    2011-05-15

    The adsorption and speciation of U(VI) was investigated on contaminated, fine grained sediment materials from the Hanford 300 area (SPP1 GWF) in simulated groundwater using cryogenic laser-induced U(VI) fluorescence spectroscopy combined with chemometric analysis. A series of reference minerals (montmorillonite, illite, Michigan chlorite, North Carolina chlorite, California clinochlore, quartz and synthetic 6-line ferrihydrite) was used for comparison that represents the mineralogical constituents of SPP1 GWF. Surface area-normalized Kd values were measured at U(VI) concentrations of 5x10-7 mol L-1 and 5x10-6 mol L-1, respectively, that displayed the following affinity series: 6-line-ferrihydrite > North Carolina chlorite ≈ California clinochlore > Michigan chlorite ≈ quartz > montmorillonite ≈ illite ≈ SPP1 GWF. Both time-resolved spectra and asynchronous two-dimensional (2D) correlation analysis of SPP1 GWF at different delay times indicated that two major adsorbed U(VI) species were present in the sediment that resembled U(VI) adsorbed on quartz and phyllosilicates. Simulations of the normalized fluorescence spectra confirmed that the speciation of SPP1 GWF was best represented by a linear combination of U(VI) adsorbed on quartz (90%) and phyllosilicates (10%). However, the fluorescence quantum yield for U(VI) adsorbed on phyllosilicates was lower than quartz and, consequently, its fractional contribution to speciation may be underestimated. Spectral comparison with literature data suggested that U(VI) exists primarily as inner-sphere U(VI) complexes with surface silanol groups on quartz while U(VI) on phyllosilicates was consistent with the formation of surface U(VI) tricarbonate complexes.

  1. Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Widengren, Jerker; Mets, Ülo; Rigler, Rudolf

    1999-12-01

    GFPs are upon excitation influenced by many different photophysical and photochemical processes effective over a very broad time scale. Much effort has been spent to investigate these processes. However, in the microsecond to millisecond time-range many processes still remain to be further characterized. This time-range can be conveniently covered by FCS, and is used here to study the photodynamical behaviour of wild-type (WT) and a F64L S65T mutant (BioST) of GFP. In addition to intersystem crossing to the triplet state, additional photophysical processes are seen, showing identical fluctuations in fluorescence to those found for a reversible photo-induced isomerization process, as well as fluctuations, not influenced by the electronic state of the chromophore unit. In the nanosecond time-range a contribution to the fluorescence correlation function is observed which can be attributed to rotational diffusion, suggesting a convenient way to measure rotational diffusion of proteins expressed with GFP on a microscopic scale.

  2. In-vitro bacterial identification using fluorescence spectroscopy with an optical fiber system

    NASA Astrophysics Data System (ADS)

    Spector, Brian C.; Werkhaven, Jay A.; Smith, Dana; Reinisch, Lou

    2000-05-01

    Acute otitis media (AOM) remains a source of significant morbidity in children. With the emergence of antibiotic resistant strains of bacteria, tympanocentesis has become an important method of bacterial identification in the setting of treatment failures. Previous studies described a prototype system for the non-invasive fluorescence identification of bacteria in vitro. We demonstrate the addition of an optical fiber to allow for the identification of a specimen distant to the spectrofluorometer. Emission spectra from three bacteria, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus were successfully obtained in vitro. This represents a necessary step prior to the study of in vivo identification of bacteria in AOM using fluorescence spectroscopy.

  3. Rapid detection of authenticity and adulteration of walnut oil by FTIR and fluorescence spectroscopy: a comparative study.

    PubMed

    Li, Bingning; Wang, Haixia; Zhao, Qiaojiao; Ouyang, Jie; Wu, Yanwen

    2015-08-15

    Fourier transform infrared (FTIR) and fluorescence spectroscopy combined with soft independent modeling of class analogies (SIMCA) and partial least square (PLS) were used to detect the authenticity of walnut oil and adulteration amount of soybean oil in walnut oil. A SIMCA model of FTIR spectra could differentiate walnut oil and other oils into separate categories; the classification limit of soybean oil in walnut oil was 10%. Fluorescence spectroscopy could differentiate oil composition by the peak position and intensity of emission spectrum without multivariate analysis. The classification limit of soybean oil adulterated in walnut oil by fluorescence spectroscopy was below 5%. The deviation of the prediction model for fluorescence spectra was lower than that for FTIR spectra. Fluorescence spectroscopy was more applicable than FTIR in the adulteration detection of walnut oil, both from the determination limit and prediction deviation.

  4. Dual time-resolved temperature-jump fluorescence and infrared spectroscopy for the study of fast protein dynamics.

    PubMed

    Davis, Caitlin M; Reddish, Michael J; Dyer, R Brian

    2017-02-02

    Time-resolved temperature-jump (T-jump) coupled with fluorescence and infrared (IR) spectroscopy is a powerful technique for monitoring protein dynamics. Although IR spectroscopy of the polypeptide amide I mode is more technically challenging, it offers complementary information because it directly probes changes in the protein backbone, whereas, fluorescence spectroscopy is sensitive to the environment of specific side chains. With the advent of widely tunable quantum cascade lasers (QCL) it is possible to efficiently probe multiple IR frequencies with high sensitivity and reproducibility. Here we describe a dual time-resolved T-jump fluorescence and IR spectrometer and its application to study protein folding dynamics. A Q-switched Ho:YAG laser provides the T-jump source for both time-resolved IR and fluorescence spectroscopy, which are probed by a QCL and Ti:Sapphire laser, respectively. The Ho:YAG laser simultaneously pumps the time-resolved IR and fluorescence spectrometers. The instrument has high sensitivity, with an IR absorbance detection limit of <0.2mOD and a fluorescence sensitivity of 2% of the overall fluorescence intensity. Using a computer controlled QCL to rapidly tune the IR frequency it is possible to create a T-jump induced difference spectrum from 50ns to 0.5ms. This study demonstrates the power of the dual time-resolved T-jump fluorescence and IR spectroscopy to resolve complex folding mechanisms by complementary IR absorbance and fluorescence measurements of protein dynamics.

  5. Constant-time 2D and 3D through-bond correlation NMR spectroscopy of solids under 60 kHz MAS

    SciTech Connect

    Zhang, Rongchun; Ramamoorthy, Ayyalusamy

    2016-01-21

    Establishing connectivity and proximity of nuclei is an important step in elucidating the structure and dynamics of molecules in solids using magic angle spinning (MAS) NMR spectroscopy. Although recent studies have successfully demonstrated the feasibility of proton-detected multidimensional solid-state NMR experiments under ultrafast-MAS frequencies and obtaining high-resolution spectral lines of protons, assignment of proton resonances is a major challenge. In this study, we first re-visit and demonstrate the feasibility of 2D constant-time uniform-sign cross-peak correlation (CTUC-COSY) NMR experiment on rigid solids under ultrafast-MAS conditions, where the sensitivity of the experiment is enhanced by the reduced spin-spin relaxation rate and the use of low radio-frequency power for heteronuclear decoupling during the evolution intervals of the pulse sequence. In addition, we experimentally demonstrate the performance of a proton-detected pulse sequence to obtain a 3D {sup 1}H/{sup 13}C/{sup 1}H chemical shift correlation spectrum by incorporating an additional cross-polarization period in the CTUC-COSY pulse sequence to enable proton chemical shift evolution and proton detection in the incrementable t{sub 1} and t{sub 3} periods, respectively. In addition to through-space and through-bond {sup 13}C/{sup 1}H and {sup 13}C/{sup 13}C chemical shift correlations, the 3D {sup 1}H/{sup 13}C/{sup 1}H experiment also provides a COSY-type {sup 1}H/{sup 1}H chemical shift correlation spectrum, where only the chemical shifts of those protons, which are bonded to two neighboring carbons, are correlated. By extracting 2D F1/F3 slices ({sup 1}H/{sup 1}H chemical shift correlation spectrum) at different {sup 13}C chemical shift frequencies from the 3D {sup 1}H/{sup 13}C/{sup 1}H spectrum, resonances of proton atoms located close to a specific carbon atom can be identified. Overall, the through-bond and through-space homonuclear/heteronuclear proximities determined from the

  6. Constant-time 2D and 3D through-bond correlation NMR spectroscopy of solids under 60 kHz MAS

    PubMed Central

    Zhang, Rongchun; Ramamoorthy, Ayyalusamy

    2016-01-01

    Establishing connectivity and proximity of nuclei is an important step in elucidating the structure and dynamics of molecules in solids using magic angle spinning (MAS) NMR spectroscopy. Although recent studies have successfully demonstrated the feasibility of proton-detected multidimensional solid-state NMR experiments under ultrafast-MAS frequencies and obtaining high-resolution spectral lines of protons, assignment of proton resonances is a major challenge. In this study, we first re-visit and demonstrate the feasibility of 2D constant-time uniform-sign cross-peak correlation (CTUC-COSY) NMR experiment on rigid solids under ultrafast-MAS conditions, where the sensitivity of the experiment is enhanced by the reduced spin-spin relaxation rate and the use of low radio-frequency power for heteronuclear decoupling during the evolution intervals of the pulse sequence. In addition, we experimentally demonstrate the performance of a proton-detected pulse sequence to obtain a 3D 1H/13C/1H chemical shift correlation spectrum by incorporating an additional cross-polarization period in the CTUC-COSY pulse sequence to enable proton chemical shift evolution and proton detection in the incrementable t1 and t3 periods, respectively. In addition to through-space and through-bond 13C/1H and 13C/13C chemical shift correlations, the 3D 1H/13C/1H experiment also provides a COSY-type 1H/1H chemical shift correlation spectrum, where only the chemical shifts of those protons, which are bonded to two neighboring carbons, are correlated. By extracting 2D F1/F3 slices (1H/1H chemical shift correlation spectrum) at different 13C chemical shift frequencies from the 3D 1H/13C/1H spectrum, resonances of proton atoms located close to a specific carbon atom can be identified. Overall, the through-bond and through-space homonuclear/heteronuclear proximities determined from the 3D 1H/13C/1H experiment would be useful to study the structure and dynamics of a variety of chemical and biological

  7. Coriolis interaction of the ν12 and 2ν10 bands of ethylene-cis-1,2-d2 (cis-C2H2D2) by high-resolution FTIR spectroscopy

    NASA Astrophysics Data System (ADS)

    Ng, L. L.; Tan, T. L.; Gabona, M. G.

    2015-10-01

    The spectrum of the A-type ν12 band of ethylene-cis-1,2-d2 (cis-C2H2D2) was recorded at an unapodized resolution of 0.0063 cm-1 in the wavenumber range of 1270-1410 cm-1. The band is perturbed through a c-type Coriolis resonance with the unobserved B-type 2ν10 band which is situated approximately 11 cm-1 below the ν12 band center. In this work, a total of 73 new infrared transitions of high J and Ka values of the ν12 band were identified and assigned for a rovibrational analysis. Finally, a total of 844 perturbed and unperturbed infrared transitions (including those previously reported) of ν12 were assigned and fitted using Watson's A-reduced Hamiltonian in the Ir representation with the inclusion of a second-order c-type Coriolis interaction term to derive a set of rovibrational constants of better accuracy for the ν12 = 1 state up to two sextic terms. Improved rotational and two quartic centrifugal distortion constants were also derived for the ν10 = 2 state of cis-C2H2D2 from the analysis of the Coriolis interaction between the two perturbing bands. The ν12 band is found to be centered at 1341.150877 ± 0.000088 cm-1 while that of 2ν10 is 1330.6360 ± 0.0113 cm-1. By fitting the infrared lines of ν12 with an rms deviation of 0.00067 cm-1, a second-order c-Coriolis coupling constant was accurately determined. A set of ground state rovibrational constants up to two sextic terms of comparable accuracy to those previously reported was also derived from a simultaneous fit of a total of 1728 ground state combination differences (GSCDs) from the infrared transitions of the present analysis and those of the ν7 band of cis-C2H2D2 together with 22 microwave transitions. The root-mean-square deviation of the GSCD fit was 0.00030 cm-1.

  8. Fluorescence excitation-emission matrix spectroscopy of vitiligo skin in vivo (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Zhao, Jianhua; Richer, Vincent; Al Jasser, Mohammed; Zandi, Soodabeh; Kollias, Nikiforos; Kalia, Sunil; Zeng, Haishan; Lui, Harvey

    2016-02-01

    Fluorescence signals depend on the intensity of the exciting light, the absorption properties of the constituent molecules, and the efficiency with which the absorbed photons are converted to fluorescence emission. The optical features and appearance of vitiligo have been explained primarily on the basis of reduced epidermal pigmentation, which results in abnormal white patches on the skin. The objective of this study is to explore the fluorescence properties of vitiligo and its adjacent normal skin using fluorescence excitation-emission matrix (EEM) spectroscopy. Thirty five (35) volunteers with vitiligo were acquired using a double-grating spectrofluorometer with excitation and emission wavelengths of 260-450 nm and 300-700 nm respectively. As expected, the most pronounced difference between the spectra obtained from vitiligo lesions compared to normally pigmented skin was that the overall fluorescence was much higher in vitiligo; these differences increased at shorter wavelengths, thus matching the characteristic spectral absorption of epidermal melanin. When comparing the fluorescence spectra from vitiligo to normal skin we detected three distinct spectral bands centered at 280nm, 310nm, and 335nm. The 280nm band may possibly be related to inflammation, whereas the 335 nm band may arise from collagen or keratin cross links. The source of the 310 nm band is uncertain; it is interesting to note its proximity to the 311 nm UV lamps used for vitiligo phototherapy. These differences are accounted for not only by changes in epidermal pigment content, but also by other optically active cutaneous biomolecules.

  9. Moving in on the Action: An Experimental Comparison of Fluorescence Excitation and Photodissociation Action Spectroscopy.

    PubMed

    Wellman, Sydney M J; Jockusch, Rebecca A

    2015-06-18

    Photodissociation action spectroscopy is often used as a proxy for measuring gas-phase absorption spectra of ions in a mass spectrometer. Although the potential discrepancy between linear optical and photodissociation spectra is generally acknowledged, direct experimental comparisons are lacking. In this work, we use a quadrupole ion trap that has been modified to enable both photodissociation and laser-induced fluorescence to assess how closely the visible photodissociation action spectrum of a fluorescent dye reflects its fluorescence excitation spectrum. Our results show the photodissociation action spectrum of gaseous rhodamine 110 is both substantially narrower and slightly red-shifted (∼120 cm(-1)) compared to its fluorescence excitation spectrum. Power dependence measurements reveal that the photodissociation of rhodamine 110 requires, on average, the absorption of three photons whereas fluorescence is a single-photon process. These differing power dependences are the key to interpreting the differences in the measured spectra. The experimental results provide much-needed quantification and insight into the differences between action spectra and linear optical spectra, and emphasize the utility of fluorescence excitation spectra to provide a more reliable benchmark for comparison with theory.

  10. Complexation between Hg(II) and biofilm extracellular polymeric substances: an application of fluorescence spectroscopy.

    PubMed

    Zhang, Daoyong; Pan, Xiangliang; Mostofa, Khan M G; Chen, Xi; Mu, Guijin; Wu, Fengchang; Liu, Jing; Song, Wenjuan; Yang, Jianying; Liu, Yanli; Fu, Qinglong

    2010-03-15

    The three-dimensional excitation emission matrix (EEM) fluorescence spectroscopy was employed to investigate the interaction of extracellular polymeric substances (EPS) from natural biofilm with Hg(II). The EEM spectra demonstrated that EPS with molecular weight over 14 kDa had two protein-like fluorescence peaks. The fluorescence intensity at both peaks was strongly dependent on the solution pH in the absence and presence of Hg(II), with the maximal fluorescence intensity at neutral pH. Fluorescence of both protein-like peaks was significantly quenched by Hg(II). The values of conditional stability constants (log K(a)=3.28-4.48) derived from modified Stern-Volmer equation are approximate to those for humic substances and dissolved organic matter (DOM), indicating that fluorescent components in EPS have strong binding capacity for Hg(II). Our findings suggest that EPS from biofilm is a class of important organic ligands for complexation with Hg(II) and may significantly affect the chemical forms, mobility, bioavailability and ecotoxicity of heavy metals in the aquatic environment.

  11. Imaging fluorescence correlation spectroscopy: nonuniform IgE distributions on planar membranes.

    PubMed Central

    Huang, Z; Thompson, N L

    1996-01-01

    Fluorescence correlation spectroscopy is useful for detecting and characterizing molecular clusters that are smaller than or approximately equal to optical resolution in size. Here, we report the development of an approach in which the pixel-to-pixel fluorescence fluctuations from a single fluorescence image are spatially autocorrelated. In these measurements, tetramethylrhodamine-labeled, anti-trinitrophenyl IgE antibodies were specifically bound to substrate-supported planar membranes composed of trinitrophenyl-aminocaproyldipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. The antibody-coated membranes were illuminated with the evanescent field from a totally internally reflected laser beam, and the fluorescence arising from the IgE-coated membranes was recorded with a cooled CCD camera. The image was corrected for the elliptical Gaussian shape of the evanescent illumination after background subtraction. The spatial autocorrelation functions of the resulting images generated two useful parameters: the extrapolated initial values, which were related to the average cluster intensity and density; and the correlation distances, which were related to the average cluster size. These parameters varied with the IgE density, and unlabeled polyclonal anti-IgE enhanced the nonuniform IgE distributions. The autocorrelation functions calculated from images of planar membranes containing fluorescently labeled lipids rather than bound, labeled IgE demonstrated that the spatial nonuniformities were prominent only in the presence of IgE. Fluorescent beads were used to demonstrate the principles and the methods. Images FIGURE 3 PMID:8785359

  12. In vivo detection of membrane protein expression using surface plasmon enhanced fluorescence spectroscopy (SPFS).

    PubMed

    Krupka, Simone S; Wiltschi, Birgit; Reuning, Ute; Hölscher, Kerstin; Hara, Masahiko; Sinner, Eva-Kathrin

    2006-08-15

    Surface plasmon enhanced fluorescence spectroscopy (SPFS) was applied for the detection of expression and functional incorporation of integral membrane proteins into plasma membranes of living cells in real time. A vesicular stomatitis virus (VSV) tagged mutant of photoreceptor bovine rhodopsin was generated for high level expression with the semliki forest virus (SFV) system. Adherent baby hamster kidney (BHK-21) cells were cultivated on fibronectin-coated gold surfaces and infected with genetically engineered virus driving the expression of rhodopsin. Using premixed fluorescently (Alexa Fluor 647) labeled anti-mouse secondary antibody and monoclonal anti-VSV primary antibody, expression of rhodopsin in BHK-21 cells was monitored by SPFS. Fluorescence enhancement by surface plasmons occurs exclusively in the close vicinity of the gold surface. Thus, only the Alexa Fluor 647 labeled antibodies binding to the VSV-tag at rhodopsin molecules exposed on the cell surface experienced fluorescence enhancement, whereas, unbound antibody molecules in the bulk solution were negligibly excited. With this novel technique, we successfully recorded an increase of fluorescence with proceeding rhodopsin expression. Thus, we were able to observe the incorporation of heterologously expressed rhodopsin in the plasma membrane of living cells in real time using a relatively simple and rapid method. We confirmed our results by comparison with conventional wide field fluorescence microscopy.

  13. Classification and characterization of beef muscles using front-face fluorescence spectroscopy.

    PubMed

    Sahar, Amna; Dufour, Eric

    2015-02-01

    The objective of this study was to evaluate the potential of fluorescence spectroscopy to identify different muscles and to predict some physicochemical and rheological parameters. Samples were taken from three muscles (Semitendinosus, Rectus abdominis and Infraspinatus) of Charolais breed. Dry matter content, fat content, protein content, texture and collagen content were determined. Moreover emission spectra were recorded in the range of 305-400nm, 340-540nm and 410-700nm by fixing the excitation wavelength at 290, 322 and 382nm, respectively. The results obtained were evaluated by partial least square discriminant analysis and partial least square regression. Results of our research work show that front-face fluorescence spectroscopy and chemometrics offer significant potential for the development of rapid and non-destructive methods for the identification and characterization of muscles.

  14. Fluorescence quantum yield of Yb3+-doped tellurite glasses determined by thermal lens spectroscopy

    NASA Astrophysics Data System (ADS)

    Lima, S. M.; Souza, A. K. R.; Langaro, A. P.; Silva, J. R.; Costa, F. B.; Moraes, J. C. S.; Figueiredo, M. S.; Santos, F. A.; Baesso, M. L.; Nunes, L. A. O.; Andrade, L. H. C.

    2017-01-01

    In this work, the combination of three different thermal lens spectroscopic methodologies was used to better determine the fluorescence quantum yield and to observe the concentration quenching in Yb3+-doped binary tellurite glasses (in mol%, 80TeO2 - 20Li2O and 80TeO2 - 20WO3). The samples were synthesized by the conventional melt-quenching method and then studied using optical spectroscopy and thermal lens spectroscopy (TLS). These characterizations enabled investigation of the radiative and nonradiative processes involved in the ytterbium doped systems. High fluorescence quantum yield was obtained for low Yb3+ doping (>90%), and in both glasses the Yb3+ presented concentration quenching mainly caused by impurities, host-ion interaction and OH- vibrations. The observations suggested that there is a possibility of doping the glasses with higher Yb concentration (>1.6 × 1021 ions/cm3) with low reduction of the quantum yield.

  15. Dynamics-based selective 2D {sup 1}H/{sup 1}H chemical shift correlation spectroscopy under ultrafast MAS conditions

    SciTech Connect

    Zhang, Rongchun; Ramamoorthy, Ayyalusamy

    2015-05-28

    Dynamics plays important roles in determining the physical, chemical, and functional properties of a variety of chemical and biological materials. However, a material (such as a polymer) generally has mobile and rigid regions in order to have high strength and toughness at the same time. Therefore, it is difficult to measure the role of mobile phase without being affected by the rigid components. Herein, we propose a highly sensitive solid-state NMR approach that utilizes a dipolar-coupling based filter (composed of 12 equally spaced 90° RF pulses) to selectively measure the correlation of {sup 1}H chemical shifts from the mobile regions of a material. It is interesting to find that the rotor-synchronized dipolar filter strength decreases with increasing inter-pulse delay between the 90° pulses, whereas the dipolar filter strength increases with increasing inter-pulse delay under static conditions. In this study, we also demonstrate the unique advantages of proton-detection under ultrafast magic-angle-spinning conditions to enhance the spectral resolution and sensitivity for studies on small molecules as well as multi-phase polymers. Our results further demonstrate the use of finite-pulse radio-frequency driven recoupling pulse sequence to efficiently recouple weak proton-proton dipolar couplings in the dynamic regions of a molecule and to facilitate the fast acquisition of {sup 1}H/{sup 1}H correlation spectrum compared to the traditional 2D NOESY (Nuclear Overhauser effect spectroscopy) experiment. We believe that the proposed approach is beneficial to study mobile components in multi-phase systems, such as block copolymers, polymer blends, nanocomposites, heterogeneous amyloid mixture of oligomers and fibers, and other materials.

  16. Analysis of some Nigerian solid mineral ores by energy-dispersive X-ray fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Obiajunwa, E. I.

    2001-11-01

    Determination of major, minor and trace elements in some Nigerian solid mineral ores by energy-dispersive X-ray fluorescence (EDXRF) spectroscopy is described. Concentration values of major, minor and trace elements for Z>18 are reported. The mineral ores studied include (i) tantalite-coloumbite minerals, (ii) bismuth minerals and (iii) lead minerals. The accuracy and precision of the technique for chemical analysis was assured by analysing the geological standards mica-Fe (biotite) and NBS 278 (obsidian).

  17. Tea quality and classification evaluation using multi-wavelength light-emitting diodes induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Feng, Chao; Liu, Xuan; Yan, Chunsheng; He, Sailing; Mei, Liang

    2013-03-01

    In this paper, we demonstrated a detective system to evaluate the quality and classification of different tea samples based on multi-wavelength LED-induced fluorescence spectroscopy. By utilizing multiple excitation wavelengths, we obtained much more physical and chemical information from the detected samples than single excitation wavelength. By utilizing principal component analysis (PCA), we extracted the dominant features of the samples to classify and characterize the tea samples.

  18. Fluorescent atom coincidence spectroscopy of extremely neutron-deficient barium isotopes

    NASA Astrophysics Data System (ADS)

    Wells, S. A.; Evans, D. E.; Griffith, J. A. R.; Eastham, D. A.; Groves, J.; Smith, J. R. H.; Tolfree, D. W. L.; Warner, D. D.; Billowes, J.; Grant, I. S.; Walker, P. M.

    1988-09-01

    Fluorescent atom coincidence spectroscopy (FACS) has been used to measure the nuclear mean square radii and moments of the extremely neutron-deficient isotopes 120-124Ba. At N=65 an abrupt change in nuclear mean square charge radii is observed which can be understood in terms of the occupation of the spin-orbit partner g7/25/2[413] neutron and g9/29/2[404] proton orbitals and the consequent enhancement of the n-p interaction.

  19. Studies of multifrequency phase-resolved fluorescence spectroscopy for spectral fingerprinting

    SciTech Connect

    McGown, L.B.

    1989-01-01

    During the first project period, we have explored several different aspects of phase-resolved fluorescence spectroscopy (PRFS) for the fingerprinting of complex samples. It should be noted that our goal is not only fingerprinting'' per se, but also includes the characterization of complex samples with respect to dynamic interactions of luminescent molecules with each other and with sample matrix constituents. Each area of investigation is discussed in the following sections.

  20. Investigation of pH-dependent photophysical properties of quantum nanocrystals by fluorescence correlation spectroscopy.

    PubMed

    Oura, Makoto; Yamamoto, Johtaro; Jin, Takashi; Kinjo, Masataka

    2017-01-23

    Quantum dot (QD) and quantum rod (QR) nanocrystals are widely used non-organic nanocrystals. Their strong fluorescence and photostability make them suitable for biomedical imaging applications. However, their pH-dependence and antibunching properties have not been studied much, especially in aqueous conditions. In this report, we used fluorescence correlation spectroscopy (FCS) with high temporal resolution to demonstrate that the fluorescent blinking and antibunching of QDs/QRs can be changed by varying the pH of their solutions. Furthermore, herein, we reported the relationship between the aggregation and antibunching relaxation time of QDs/QRs for the first time. The findings of this study suggest that FCS can be used to discover novel environmental indicators via observing nanosecond and microsecond phenomena.

  1. Applying Fluorescence Correlation Spectroscopy to Investigate Peptide-Induced Membrane Disruption.

    PubMed

    Kristensen, Kasper; Henriksen, Jonas R; Andresen, Thomas L

    2017-01-01

    There is considerable interest in understanding the interactions of antimicrobial peptides with phospholipid membranes. Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that can be used to gain insight into these interactions. Specifically, FCS can be used to quantify leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles, thereby providing a tool for estimating the size of peptide-induced membrane disruptions. If fluorescently labeled lipids are incorporated into the membranes of the vesicles, FCS can also be used to obtain information about whether leakage occurs due to localized membrane perturbations or global membrane destabilization. Here, we outline a detailed step-by-step protocol on how to optimally implement an FCS-based leakage assay. To make the protocol easily accessible to other researchers, it has been supplemented with a number of practical tips and tricks.

  2. Fluorescence spectroscopy and molecular simulation on the interaction of caffeic acid with human serum albumin.

    PubMed

    Xiang, Yuhong; Duan, Lili; Ma, Qiang; Lv, Zizheng; Ruohua, Zhu; Zhang, Zhuoyong

    2016-12-01

    Fluorescence spectroscopy and molecular simulation were explored to study the interaction between caffeic acid and human serum albumin (HSA). The experimental results indicated that the fluorescence quenching mechanism between caffeic acid and HSA is a static quenching, which was proved again by the analysis of fluorescence lifetime by time-correlated single photon counting. The binding process is spontaneous and the hydrophobic force is the main force between caffeic acid and HSA. In addition, the binding of caffeic acid to HSA was modeled by molecular dynamics simulations. The root mean square deviations, root mean square fluctuations, radius of gyration and the number of hydrogen bonds of the molecular dynamic (MD) simulation process were analyzed. Both experimental and modeling results demonstrated strong binding between HSA and caffeic acid. HSA had a slight conformational change when it binds with caffeic acid. The obtained information is useful for HSA drug design. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Polarization-dependent fluorescence correlation spectroscopy for studying structural properties of proteins in living cell

    PubMed Central

    Oura, Makoto; Yamamoto, Johtaro; Ishikawa, Hideto; Mikuni, Shintaro; Fukushima, Ryousuke; Kinjo, Masataka

    2016-01-01

    Rotational diffusion measurement is predicted as an important method in cell biology because the rotational properties directly reflect molecular interactions and environment in the cell. To prove this concept, polarization-dependent fluorescence correlation spectroscopy (pol-FCS) measurements of purified fluorescent proteins were conducted in viscous solution. With the comparison between the translational and rotational diffusion coefficients obtained from pol-FCS measurements, the hydrodynamic radius of an enhanced green fluorescent protein (EGFP) was estimated as a control measurement. The orientation of oligomer EGFP in living cells was also estimated by pol-FCS and compared with Monte Carlo simulations. The results of this pol-FCS experiment indicate that this method allows an estimation of the molecular orientation using the characteristics of rotational diffusion. Further, it can be applied to analyze the degree of molecular orientation and multimerization or detection of tiny aggregation of aggregate-prone proteins. PMID:27489044

  4. In vivo detection of epileptic brain tissue using static fluorescence and diffuse reflectance spectroscopy

    NASA Astrophysics Data System (ADS)

    Yadav, Nitin; Bhatia, Sanjiv; Ragheb, John; Mehta, Rupal; Jayakar, Prasanna; Yong, William; Lin, Wei-Chiang

    2013-02-01

    Diffuse reflectance and fluorescence spectroscopy are used to detect histopathological abnormalities of an epileptic brain in a human subject study. Static diffuse reflectance and fluorescence spectra are acquired from normal and epileptic brain areas, defined by electrocorticography (ECoG), from pediatric patients undergoing epilepsy surgery. Biopsy specimens are taken from the investigated sites within an abnormal brain. Spectral analysis reveals significant differences in diffuse reflectance spectra and the ratio of fluorescence and diffuse reflectance spectra from normal and epileptic brain areas defined by ECoG and histology. Using these spectral differences, tissue classification models with accuracy above 80% are developed based on linear discriminant analysis. The differences between the diffuse reflectance spectra from the normal and epileptic brain areas observed in this study are attributed to alterations in the static hemodynamic characteristics of an epileptic brain, suggesting a unique association between the histopathological and the hemodynamic abnormalities in an epileptic brain.

  5. Optical phantoms with variable properties and geometries for diffuse and fluorescence optical spectroscopy

    NASA Astrophysics Data System (ADS)

    Leh, Barbara; Siebert, Rainer; Hamzeh, Hussein; Menard, Laurent; Duval, Marie-Alix; Charon, Yves; Abi Haidar, Darine

    2012-10-01

    Growing interest in optical instruments for biomedical applications has increased the use of optically calibrated phantoms. Often associated with tissue modeling, phantoms allow the characterization of optical devices for clinical purposes. Fluorescent gel phantoms have been developed, mimicking optical properties of healthy and tumorous brain tissues. Specific geometries of dedicated molds offer multiple-layer phantoms with variable thicknesses and monolayer phantoms with cylindrical inclusions at various depths and diameters. Organic chromophores are added to allow fluorescence spectroscopy. These phantoms are designed to be used with 405 nm as the excitation wavelength. This wavelength is then adapted to excite large endogenous molecules. The benefits of these phantoms in understanding fluorescence tissue analysis are then demonstrated. In particular, detectability aspects as a function of geometrical and optical parameters are presented and discussed.

  6. Fluorescence spectroscopy for assessment of liver transplantation grafts concerning graft viability and patient survival

    NASA Astrophysics Data System (ADS)

    Vollet Filho, José D.; da Silveira, Marina R.; Castro-e-Silva, Orlando; Bagnato, Vanderlei S.; Kurachi, Cristina

    2015-06-01

    Evaluating transplantation grafts at harvest is essential for its success. Laser-induced fluorescence spectroscopy (LIFS) can help monitoring changes in metabolic/structural conditions of tissue during transplantation. The aim of the present study is to correlate LIFSobtained spectra of human hepatic grafts during liver transplantation with post-operative patients' mortality rate and biochemical parameters, establishing a method to exclude nonviable grafts before implantation. Orthotopic liver transplantation, piggyback technique was performed in 15 patients. LIFS was performed under 408nm excitation. Collection was performed immediately after opening donor's abdominal cavity, after cold perfusion, end of back-table period, and 5 min and 1 h after warm perfusion at recipient. Fluorescence information was compared to lactate, creatinine, bilirubin and INR levels and to survival status. LIFS was sensitive to liver changes during transplantation stages. Study-in-progress; initial results indicate correlation between fluorescence and life/death status of patients.

  7. Investigation of the interaction of pepsin with ionic liquids by using fluorescence spectroscopy.

    PubMed

    Fan, Yunchang; Zhang, Sheli; Wang, Qiang; Li, Junhai; Fan, Haotian; Shan, Dongkai

    2013-06-01

    The molecular mechanism of the interaction between pepsin and two typical ionic liquids (ILs), 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) and 1-octyl-3-methylimidazolium chloride ([C8mim]Cl), was investigated with fluorescence spectroscopy, ultraviolet absorption, and circular dichroism spectroscopy at a pH value of 1.6. The results suggest that ILs could quench the intrinsic fluorescence of pepsin, probably via a dynamic quenching mechanism. The fluorescence quenching constants were determined by employing the classic Stern-Volmer equation. The constant values are very small, indicating that only a very weak interaction between ILs and pepsin exists. The Gibbs free-energy change, enthalpy change (ΔH), and entropy change (ΔS) during the interaction of pepsin and ILs were estimated. Positive values of ΔH and ΔS indicate that the interaction between ILs and pepsin is mainly driven by hydrophobic interaction. Synchronous and three-dimensional fluorescence spectra demonstrate that the addition of ILs (0-0.20 mol L(-1) for each IL) does not bring apparent changes to the microenvironments of tyrosine and tryptophan residues. Activity experiments show that the activity of pepsin is concentration dependent; higher concentrations of ILs (>0.22 mol L(-1) for [C8mim]Cl and >0.30 mol L(-1) for [C4mim]Cl) cause the remarkable reduction of enzyme activity. The presence of ILs also does not improve the thermal stability of pepsin.

  8. Evanescent field in surface plasmon resonance and surface plasmon field-enhanced fluorescence spectroscopies.

    PubMed

    Ekgasit, Sanong; Thammacharoen, Chuchaat; Yu, Fang; Knoll, Wolfgang

    2004-04-15

    The highly sensitive nature of surface plasmon resonance (SPR) spectroscopy and surface plasmon field-enhanced fluorescence spectroscopy (SPFS) are governed by the strong surface plasmon resonance-generated evanescent field at the metal/dielectric interface. The greatest evanescent field amplitude at the interface and the maximum attenuation of the reflectance are observed when a nonabsorbing dielectric is employed. An absorbing dielectric decreases the evanescent field enhancement at the interface. The SPR curve of an absorbing dielectric is characterized by a greater reflectance minimum and a broader curve, as compared to those of the nonabsorbing dielectric with the same refractive index. For a weakly absorbing dielectric, such as nanometer-thick surface-confined fluorophores, the absorption is too small to induce a significant change in the SPR curve. However, the presence of a minute amount of the fluorophore can be detected by the highly sensitive SPFS. The angle with the maximum fluorescence intensity of an SPFS curve is always smaller than the resonance angle of the corresponding SPR curve. This discrepancy is due to the differences of evanescent field distributions and their decay characteristics within the metal film and the dielectric medium. The fluorescence intensity in an SPFS curve can be expressed in terms of the evanescent field amplitude. Excellent correlations between the experimentally measured fluorescence intensities and the evanescent field amplitudes are observed.

  9. Spoilage of foods monitored by native fluorescence spectroscopy with selective excitation wavelength

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Wang, Wubao; Alfano, Robert R.

    2015-03-01

    The modern food processing and storage environments require the real-time monitoring and rapid microbiological testing. Optical spectroscopy with selective excitation wavelengths can be the basis of a novel, rapid, reagent less, noncontact and non-destructive technique for monitoring the food spoilage. The native fluorescence spectra of muscle foods stored at 2-4°C (in refrigerator) and 20-24°C (in room temperature) were measured as a function of time with a selective excitation wavelength of 340nm. The contributions of the principal molecular components to the native fluorescence spectra of meat were measured spectra of each fluorophore: collagen, reduced nicotinamide adenine dinucleotide (NADH), and flavin. The responsible components were extracted using a method namely Multivariate Curve Resolution with Alternating Least-Squares (MCR-ALS). The native fluorescence combined with MCR-ALS can be used directly on the surface of meat to produce biochemically interpretable "fingerprints", which reflects the microbial spoilage of foods involved with the metabolic processes. The results show that with time elapse, the emission from NADH in meat stored at 24°C increases much faster than that at 4°C. This is because multiplying of microorganisms and catabolism are accompanied by the generation of NADH. This study presents changes of relative content of NADH may be used as criterion for detection of spoilage degree of meat using native fluorescence spectroscopy.

  10. Applications of Fluorescence Spectroscopy for dissolved organic matter characterization in wastewater treatment plants

    NASA Astrophysics Data System (ADS)

    Goffin, Angélique; Guérin, Sabrina; Rocher, Vincent; Varrault, Gilles

    2016-04-01

    Dissolved organic matter (DOM) influences wastewater treatment plants efficiency (WTTP): variations in its quality and quantity can induce a foaming phenomenon and a fouling event inside biofiltration processes. Moreover, in order to manage denitrification step (control and optimization of the nitrate recirculation), it is important to be able to estimate biodegradable organic matter quantity before biological treatment. But the current methods used to characterize organic matter quality, like biological oxygen demand are laborious, time consuming and sometimes not applicable to directly monitor organic matter in situ. In the context of MOCOPEE research program (www.mocopee.com), this study aims to assess the use of optical techniques, such as UV-Visible absorbance and more specifically fluorescence spectroscopy in order to monitor and to optimize process efficiency in WWTP. Fluorescence excitation-emission matrix (EEM) spectroscopy was employed to prospect the possibility of using this technology online and in real time to characterize dissolved organic matter in different effluents of the WWTP Seine Centre (240,000 m3/day) in Paris, France. 35 sewage water influent samples were collected on 10 days at different hours. Data treatment were performed by two methods: peak picking and parallel factor analysis (PARAFAC). An evolution of DOM quality (position of excitation - emission peaks) and quantity (intensity of fluorescence) was observed between the different treatment steps (influent, primary treatment, biological treatment, effluent). Correlations were found between fluorescence indicators and different water quality key parameters in the sewage influents. We developed different multivariate linear regression models in order to predict a variety of water quality parameters by fluorescence intensity at specific excitation-emission wavelengths. For example dissolved biological oxygen demand (r2=0,900; p<0,0001) and ammonium concentration (r2=0,898; p<0

  11. The Coriolis-interacting ν6 and ν4 bands of ethylene-cis-1,2-d2 (cis-C2H2D2) by high-resolution synchrotron Fourier transform infrared (FTIR) spectroscopy

    NASA Astrophysics Data System (ADS)

    Tan, T. L.; Gabona, M. G.; Wong, Andy; Appadoo, Dominique R. T.; McNaughton, Don

    2016-11-01

    The infrared spectrum of the ν6 band of ethylene-cis-1,2-d2 (cis-C2H2D2) was recorded at the Australian Synchrotron in the 980-1100 cm-1 region at an unapodized resolution of 0.00096 cm-1. Some of the transitions of the ν6 band centered at 1039.768335(30) cm-1 were perturbed by the upper energy levels of the infrared inactive ν4 band at 980.364(24) cm-1 by an a-type Coriolis interaction. Rovibrational analysis of a total of 941 unperturbed and perturbed infrared transitions of the ν6 band was carried out using an asymmetric rotor fitting program based on the Watson's A-reduced Hamiltonian in the Ir representation to derive up to 2 sextic constants for the ν6 = 1 state and 3 rotational constants (A, B, and C) for the ν4 = 1 state with a rms deviation of 0.00028 cm-1. From the perturbed analysis, the a-type Coriolis resonance parameter Z6,4a for the ν6 and ν4 interacting bands was determined to be 0.5249(14) cm-1. The band center and the rotational constants of the ν6 = 1 state were found to agree within 1% to the calculated values using B3LYP/cc-pVTZ and MP2/cc-pVTZ levels of theory. Furthermore, the a-type Coriolis coupling constant of these two bands derived from this work were compared to those experimentally determined previously and presently calculated.

  12. Disassembly of structurally modified viral nanoparticles: characterization by fluorescence correlation spectroscopy.

    PubMed

    Toivola, Jouni; Gilbert, Leona; Michel, Patrik; White, Daniel; Vuento, Matti; Oker-Blom, Christian

    2005-12-01

    Analysis of the breakdown products of engineered viral particles can give useful information on the particle structure. We used various methods to breakdown both a recombinant enveloped virus and virus-like particles (VLPs) from two non-enveloped viruses and analysed the resulting subunits by fluorescence correlation spectroscopy (FCS). Analysis of the enveloped baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), displaying the green fluorescent protein (GFP) fused to its envelope protein gp64 was performed in the presence and absence of 5 mM SDS and 25 mM DTT. Without treatment, the viral particle showed a diffusion time of 3.3 ms. In the presence of SDS, fluorescent subunits with diffusion times of 0.2 ms were observed. Additional treatment with DTT caused a drop in the diffusion time to 0.1 ms. Changes in the amplitude of the autocorrelation function suggested a 3-fold increase in fluorescent particle number when viral particles were treated with SDS, and a further 1.5-fold increase with additional treatment with DTT. Thus, the data showed that an average of 4.5 molecules of gp64-GFP was incorporated in the membrane of the modified baculovirus. Further, this suggests that each fluorescent gp64 trimer carries on average 1.5 fluorescent units. Similar experiments were carried out with two non-enveloped fluorescent virus-like particles (fVLPs) that displayed enhanced green fluorescent protein (EGFP). These, fVLPs of canine and human B19 parvoviruses were treated with 6 M urea and 5 mM SDS, respectively. Correspondingly, the original hydrodynamic radii of 17 and 14 nm were reduced to 9 and 5 nm after treatment. Here, the change in the amplitude of the autocorrelation curve suggested a 10-fold increase in particle number when viral particles of CPV were treated with 6 M urea at 50 degrees C for 10 min. For EGFP-B19, there was a decrease in the amplitude, accompanied by a 9-fold increase in the number of fluorescent units with SDS treatment

  13. Analyzing pH-induced changes in a myofibril model system with vibrational and fluorescence spectroscopy.

    PubMed

    Andersen, Petter Vejle; Veiseth-Kent, Eva; Wold, Jens Petter

    2017-03-01

    The decline of pH and ultimate pH in meat postmortem greatly influences meat quality (e.g. water holding capacity). Four spectroscopic techniques, Raman, Fourier transform infrared (FT-IR), near infrared (NIR) and fluorescence spectroscopy, were used to study protein and amino acid modifications to determine pH-related changes in pork myofibril extracts at three different pH-levels, 5.3, 5.8 and 6.3. Protonation of side-chain carboxylic acids of aspartic and glutamic acid and changes in secondary structure, mainly the amide I-III peaks, were the most important features identified by Raman and FT-IR spectroscopy linked to changes in pH. Fluorescence spectroscopy identified tryptophan interaction with the molecular environment as the most important contributor to changes in the spectra. NIR spectroscopy gave no significant contributions to interpreting protein structure related to pH. Results from our study are useful for interpreting spectroscopic data from meat where pH is an important variable.

  14. A high-resolution large-acceptance analyzer for X-ray fluorescence and Raman spectroscopy

    SciTech Connect

    Bergmann, Uwe; Cramer, Stephen P.

    2001-08-02

    A newly designed multi-crystal X-ray spectrometer and its applications in the fields of X-ray fluorescence and X-ray Raman spectroscopy are described. The instrument is based on 8 spherically curved Si crystals, each with a 3.5 inch diameter form bent to a radius of 86 cm. The crystals are individually aligned in the Rowland geometry capturing a total solid angle of 0.07 sr. The array is arranged in a way that energy scans can be performed by moving the whole instrument, rather than scanning each crystal by itself. At angles close to back scattering the energy resolution is between 0.3 and 1 eV depending on the beam dimensions at the sample. The instrument is mainly designed for X-ray absorption and fluorescence spectroscopy of transition metals in dilute systems such as metalloproteins. First results of the Mn K{beta} (3p -> 1s) emission in photosystem II are shown. An independent application of the instrument is the technique of X-ray Raman spectroscopy which can address problems similar to those in traditional soft X-ray absorption spectroscopies, and initial results are presented.

  15. Construction, figures of merit, and testing of a single-cell fluorescence excitation spectroscopy system

    PubMed Central

    Hill, Laura S.; Richardson, Tammi L.; Profeta, Luisa T. M.; Shaw, Timothy J.; Hintz, Christopher J.; Twining, Benjamin S.; Lawrenz, Evelyn; Myrick, Michael L.

    2010-01-01

    Characterization of phytoplankton community composition is critical to understanding the ecology and biogeochemistry of the oceans. One approach to taxonomic characterization takes advantage of differing pigmentation between algal taxa and thus differences in fluorescence excitation spectra. Analyses of bulk water samples, however, may be confounded by interference from chromophoric dissolved organic matter or suspended particulate matter. Here, we describe an instrument that uses a laser trap based on a Nikon TE2000-U microscope to position individual phytoplankton cells for confocal fluorescence excitation spectroscopy, thus avoiding interference from the surrounding medium. Quantitative measurements of optical power give data in the form of photons emitted per photon of exposure for an individual phytoplankton cell. Residence times for individual phytoplankton in the instrument can be as long as several minutes with no substantial change in their fluorescence excitation spectra. The laser trap was found to generate two-photon fluorescence from the organisms so a modification was made to release the trap momentarily during data acquisition. Typical signal levels for an individual cell are in the range of 106 photons∕s of fluorescence using a monochromated 75 W Xe arc lamp excitation source with a 2% transmission neutral density filter. PMID:20113077

  16. Identifying the origins of microbially derived aquatic DOM using fluorescence spectroscopy.

    NASA Astrophysics Data System (ADS)

    Fox, Bethany; Thorn, Robin; Anesio, Alexandre; Reynolds, Darren

    2016-04-01

    Dissolved organic matter (DOM) in aquatic systems is an essential support of the microbial population and, therefore, of the entire aquatic ecosystem. Aquatic DOM is also key for global biogeochemical cycling of nutrients and connects land processes to the marine environment via hydrological transportation. There have been multiple advances in technological assessments of the characteristics of aquatic DOM, with spectroscopy becoming widely used. The extensive use of benchtop spectroscopic instruments has led to the development of in situ sensors, improving the spatiotemporal scale of data acquisition. Whilst this has greatly improved understanding of DOM characteristics and patterns, there are still unknown variables, parameters and interactions of DOM within the aquatic environment. In particular, the interactions of aquatic DOM with the microbial population is still mostly unidentified. It is generally accepted that certain DOM fluorescence regions are autochthonous and microbially derived, such as "peak T" fluorescence. However, the origins and metabolic pathways involved in the production and release of these fluorescent molecules is, as yet, not definitively known. Our work focuses on the identification of these metabolic pathways from whence this microbially derived DOM originates. The most recent stage of the research has utilised traditional microbiological techniques, such as growth curves and chemostat experiments, alongside DOM fluorescence spectroscopic analysis and flow cytometry. The information gained regarding the microbial production and processing of DOM is central for the development of novel in situ fluorescence technology, the ultimate aim of this project.

  17. Applications of fluorescence spectroscopy for predicting percent wastewater in an urban stream

    USGS Publications Warehouse

    Goldman, Jami H.; Rounds, Stewart A.; Needoba, Joseph A.

    2012-01-01

    Dissolved organic carbon (DOC) is a significant organic carbon reservoir in many ecosystems, and its characteristics and sources determine many aspects of ecosystem health and water quality. Fluorescence spectroscopy methods can quantify and characterize the subset of the DOC pool that can absorb and re-emit electromagnetic energy as fluorescence and thus provide a rapid technique for environmental monitoring of DOC in lakes and rivers. Using high resolution fluorescence techniques, we characterized DOC in the Tualatin River watershed near Portland, Oregon, and identified fluorescence parameters associated with effluent from two wastewater treatment plants and samples from sites within and outside the urban region. Using a variety of statistical approaches, we developed and validated a multivariate linear regression model to predict the amount of wastewater in the river as a function of the relative abundance of specific fluorescence excitation/emission pairs. The model was tested with independent data and predicts the percentage of wastewater in a sample within 80% confidence. Model results can be used to develop in situ instrumentation, inform monitoring programs, and develop additional water quality indicators for aquatic systems.

  18. Enhanced energy transfer in respiratory-deficient endothelial cells probed by microscopic fluorescence excitation spectroscopy

    NASA Astrophysics Data System (ADS)

    Schneckenburger, Herbert; Gschwend, Michael H.; Bauer, Manfred; Strauss, Wolfgang S. L.; Steiner, Rudolf W.

    1996-12-01

    Mitochondrial malfunction may be concomitant with changes of the redox states of the coenzymes nicotinamide adenine dinucleotide (NAD+/NADH), as well as flavin.mononucleotide or dinucleotide. The intrinsic fluorescence of these coenzymes was therefore proposed to be a measure of malfunction. Since mitochondrial fluorescence is strongly superposed by autofluorescence from various cytoplasmatic fluorophores, cultivated endothelial cells were incubated with the mitochondrial marker rhodamine 123 (R123), and after excitation of flavin molecules, energy transfer to R123 was investigated. Due to spectral overlap of flavin and R123 fluorescence, energy transfer flavin yields R123 could not be detected from their emission spectra. Therefore, the method of microscopic fluorescence excitation spectroscopy was established. When detecting R123 fluorescence, excitation maxima at 370 - 390 nm and 420-460 nm were assigned to flavins, whereas a pronounced excitation band at 465 - 490 nm was attributed to R123. Therefore, excitation at 475 nm reflected the intracellular concentration of R123, whereas excitation at 385 nm reflected flavin excitation with a subsequent energy transfer to R123 molecules. An enhanced energy transfer after inhibition of specific enzyme complexes of the respiratory chain is discussed in the present article.

  19. Effect of dielectric spacer thickness on signal intensity of surface plasmon field-enhanced fluorescence spectroscopy.

    PubMed

    Murakami, Takashi; Arima, Yusuke; Toda, Mitsuaki; Takiguchi, Hiromi; Iwata, Hiroo

    2012-02-15

    Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) combines enhanced field platform and fluorescence detection. Its advantages are the strong intensity of the electromagnetic field and the high signal/noise (S/N) ratio due to the localized evanescent field at the water/metal interface. However, the energy transfer from the fluorophore to the metal surface diminishes the fluorescence intensity, and this reduces the sensitivity. In this study, we tested whether polystyrene (PSt) could act as a dielectric layer to suppress the energy transfer from the fluorophore to the metal surface. We hypothesized that this would improve the sensitivity of SPFS-based immunoassays. We used α-fetoprotein (AFP) as a model tumor biomarker in the sandwich-type immunoassay. We determined the relationship between fluorescent signal intensity and PSt layer thickness and compared this to theoretical predictions. We found that the fluorescence signal increased by optimally controlling the thickness of the PSt layer. Our results indicated that the SPFS-based immunoassay is a promising clinical diagnostic tool for quantitatively determining the concentrations of low-level biomarkers in blood samples.

  20. On-chip integrated lensless fluorescence microscopy/spectroscopy module for cell-based sensors

    NASA Astrophysics Data System (ADS)

    Li, Wei; Knoll, Thorsten; Sossalla, Adam; Bueth, Heiko; Thielecke, Hagen

    2011-03-01

    The integration of a fluorescence microscopy/spectroscopy module in cell-based lab-on-a-chip systems is of high interest for applications in cell-based diagnostics and substance evaluation in situ. We present an on-chip integrated lensless fluorescence imaging module applying the principle of contact/proximate optical lithography. The pixel resolution is comparable with a 4 x objective microscope. The module can be used for morphology and fluorescence imaging of mammalian cells (15 - 20 μm) as well as for testing the concentration of a fluorescent substance. The biological samples or solutions are sustained in disposable sterilized microfluidic chips with 1 μm thick silicon nitride (Si3N4) membranes. These chips are assembled on the surface of a 5 megapixel colored CMOS image sensor array with 1.75 μm pixel size, which is coated with an additional interference filter. Each culturing chip consists of a MEMS cavity chip and a PDMS microfluidic interface. The surface of the CMOS image sensor is smoothened using SU-8 photoresist spin-coating for a commercial grade interference filter (optical density >= 5) coating by Plasma-Ion Assisted Deposition thereafter. The function is demonstrated by primary imaging results of the non-/fluorescent mammalian cells/microspheres as well as by differentiating different concentrations of FITC solutions.

  1. Time-resolved fluorescence polarization spectroscopy of visible and near infrared dyes in picosecond dynamics

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Alfano, Robert R.

    2015-03-01

    Near-infrared (NIR) dyes absorb and emit light within the range from 700 to 900 nm have several benefits in biological studies for one- and/or two-photon excitation for deeper penetration of tissues. These molecules undergo vibrational and rotational motion in the relaxation of the excited electronic states, Due to the less than ideal anisotropy behavior of NIR dyes stemming from the fluorophores elongated structures and short fluorescence lifetime in picosecond range, no significant efforts have been made to recognize the theory of these dyes in time-resolved polarization dynamics. In this study, the depolarization of the fluorescence due to emission from rotational deactivation in solution will be measured with the excitation of a linearly polarized femtosecond laser pulse and a streak camera. The theory, experiment and application of the ultrafast fluorescence polarization dynamics and anisotropy are illustrated with examples of two of the most important medical based dyes. One is NIR dye, namely Indocyanine Green (ICG) and is compared with Fluorescein which is in visible range with much longer lifetime. A set of first-order linear differential equations was developed to model fluorescence polarization dynamics of NIR dye in picosecond range. Using this model, the important parameters of ultrafast polarization spectroscopy were identified: risetime, initial time, fluorescence lifetime, and rotation times.

  2. Characterization of dissolved organic matter in fogwater by excitation-emission matrix fluorescence spectroscopy

    USGS Publications Warehouse

    Birdwell, J.E.; Valsaraj, K.T.

    2010-01-01

    Dissolved organic matter (DOM) present in fogwater samples collected in southeastern Louisiana and central-eastern China has been characterized using excitation-emission matrix fluorescence spectroscopy. The goal of the study was to illustrate the utility of fluorescence for obtaining information on the large fraction of organic carbon in fogwaters (typically >40% by weight) that defies characterization in terms of specific chemical compounds without the difficulty inherent in obtaining sufficient fogwater volume to isolate DOM for assessment using other spectroscopic and chemical analyses. Based on the findings of previous studies using other characterization methods, it was anticipated that the unidentified organic carbon fraction would have characteristic peaks associated with humic substances and fluorescent amino acids. Both humic- and protein-like fluorophores were observed in the fogwater spectra and fluorescence-derived indices for the fogwater had similar values to those of soil and sediment porewater. Greater biological character was observed in samples with higher organic carbon concentrations. Fogwaters are shown to contain a mixture of terrestrially- and microbially-derived fluorescent organic material, which is expected to be derived from an array of different sources, such as suspended soil and dust particles, biogenic emissions and organic substances generated by atmospheric processes. The fluorescence results indicate that much of the unidentified organic carbon present in fogwater can be represented by humic-like and biologically-derived substances similar to those present in other aquatic systems, though it should be noted that fluorescent signatures representative of DOM produced by atmospheric processing of organic aerosols may be contributing to or masked by humic-like fluorophores. ?? 2010.

  3. A comparative evaluation of Raman and fluorescence spectroscopy for optical diagnosis of oral neoplasia

    NASA Astrophysics Data System (ADS)

    Majumder, S. K.; Krishna, H.; Sidramesh, M.; Chaturvedi, P.; Gupta, P. K.

    2011-08-01

    We report the results of a comparative evaluation of in vivo fluorescence and Raman spectroscopy for diagnosis of oral neoplasia. The study carried out at Tata Memorial Hospital, Mumbai, involved 26 healthy volunteers and 138 patients being screened for neoplasm of oral cavity. Spectral measurements were taken from multiple sites of abnormal as well as apparently uninvolved contra-lateral regions of the oral cavity in each patient. The different tissue sites investigated belonged to one of the four histopathology categories: 1) squamous cell carcinoma (SCC), 2) oral sub-mucous fibrosis (OSMF), 3) leukoplakia (LP) and 4) normal squamous tissue. A probability based multivariate statistical algorithm utilizing nonlinear Maximum Representation and Discrimination Feature for feature extraction and Sparse Multinomial Logistic Regression for classification was developed for direct multi-class classification in a leave-one-patient-out cross validation mode. The results reveal that the performance of Raman spectroscopy is considerably superior to that of fluorescence in stratifying the oral tissues into respective histopathologic categories. The best classification accuracy was observed to be 90%, 93%, 94%, and 89% for SCC, SMF, leukoplakia, and normal oral tissues, respectively, on the basis of leave-one-patient-out cross-validation, with an overall accuracy of 91%. However, when a binary classification was employed to distinguish spectra from all the SCC, SMF and leukoplakik tissue sites together from normal, fluorescence and Raman spectroscopy were seen to have almost comparable performances with Raman yielding marginally better classification accuracy of 98.5% as compared to 94% of fluorescence.

  4. A comparative evaluation of Raman and fluorescence spectroscopy for optical diagnosis of oral neoplasia

    NASA Astrophysics Data System (ADS)

    Majumder, S. K.; Krishna, H.; Sidramesh, M.; Chaturvedi, P.; Gupta, P. K.

    2010-12-01

    We report the results of a comparative evaluation of in vivo fluorescence and Raman spectroscopy for diagnosis of oral neoplasia. The study carried out at Tata Memorial Hospital, Mumbai, involved 26 healthy volunteers and 138 patients being screened for neoplasm of oral cavity. Spectral measurements were taken from multiple sites of abnormal as well as apparently uninvolved contra-lateral regions of the oral cavity in each patient. The different tissue sites investigated belonged to one of the four histopathology categories: 1) squamous cell carcinoma (SCC), 2) oral sub-mucous fibrosis (OSMF), 3) leukoplakia (LP) and 4) normal squamous tissue. A probability based multivariate statistical algorithm utilizing nonlinear Maximum Representation and Discrimination Feature for feature extraction and Sparse Multinomial Logistic Regression for classification was developed for direct multi-class classification in a leave-one-patient-out cross validation mode. The results reveal that the performance of Raman spectroscopy is considerably superior to that of fluorescence in stratifying the oral tissues into respective histopathologic categories. The best classification accuracy was observed to be 90%, 93%, 94%, and 89% for SCC, SMF, leukoplakia, and normal oral tissues, respectively, on the basis of leave-one-patient-out cross-validation, with an overall accuracy of 91%. However, when a binary classification was employed to distinguish spectra from all the SCC, SMF and leukoplakik tissue sites together from normal, fluorescence and Raman spectroscopy were seen to have almost comparable performances with Raman yielding marginally better classification accuracy of 98.5% as compared to 94% of fluorescence.

  5. Changes in fluorescent dissolved organic matter upon interaction with anionic surfactant as revealed by EEM-PARAFAC and two dimensional correlation spectroscopy.

    PubMed

    Maqbool, Tahir; Hur, Jin

    2016-10-01

    Surfactants are present in significant amounts in both domestic and industrial wastewater, which may interact with dissolved organic matter (DOM). The present study investigated the interactions of sodium dodecyl sulfate (SDS) with three different DOM solutions, including bovine serum albumin (BSA), humic acid (HA), and the mixture of the two (BSA-HA), based on two advanced spectroscopic tools: excitation emission matrix (EEM) combined with parallel factor analysis (EEM-PARAFAC) and two dimensional correlation spectroscopy (2D-COS). The responses of two protein-like components to the addition of SDS differed depending the presence and the absence of HA. A decreasing and an increasing trend was observed for tryptophan-like (C1) and tyrosine-like (C2) components, respectively, in the BSA solution, while the BSA-HA mixture exhibited increasing fluorescence trends for both protein-like components. The conflicting results suggest that HA plays a secondary role in the protein-SDS interactions. No interaction between the SDS and humic-like component was found. 2D-COS combined with fluorescence spectra demonstrated that the protein-SDS interaction occurred on the order of C2 > C1 for the BSA solution but C1 > C2 for the BSA-HA mixture. Analyses of Scatchard plots confirmed the sequential order interpreted from 2D-COS, showing consistent trends in the binding constants. However, the presence of HA affected the protein-SDS interactions in different manners for C1 and C2, enhancing and reducing the binding constants, respectively. Circular dichroism spectra confirmed the occurrence of conformational changes in BSA with SDS. EEM-PARAFAC and 2D-COS successfully explained different interactions of surfactant with protein-like components in the presence of HA.

  6. Multiphoton microscopy, fluorescence lifetime imaging and optical spectroscopy for the diagnosis of neoplasia

    NASA Astrophysics Data System (ADS)

    Skala, Melissa Caroline

    2007-12-01

    the ultraviolet to visible wavelength range indicated that the most diagnostic optical signals originate from sub-surface tissue layers. Optical properties extracted from these spectroscopy measurements showed a significant decrease in the hemoglobin saturation, absorption coefficient, reduced scattering coefficient and fluorescence intensity (at 400 nm excitation) in neoplastic compared to normal tissues. The results from these studies indicate that multiphoton microscopy and optical spectroscopy can non-invasively provide information on tissue structure and function in vivo that is related to tissue pathology.

  7. Fluorescence spectroscopy of teeth and bones of rats to assess demineralization: In vitro, in vivo and ex vivo studies.

    PubMed

    Paolillo, Fernanda Rossi; Romano, Renan Arnon; de Matos, Luciana; Rodrigues, Phamilla Gracielli Sousa; Panhóca, Vitor Hugo; Martin, Airton Abrahão; Soares, Luis Eduardo Silva; de Castro Neto, Jarbas Caiado; Bagnato, Vanderlei Salvador

    2016-12-01

    This study investigated the effects of demineralization on teeth and bones evaluated by fluorescence spectroscopy and micro energy-dispersive X-ray fluorescence spectrometry (μ-EDXRF) in rats. For in vitro study, 20 teeth of Wistar rats were removed and decalcified to evaluate fluorescence. For in vivo study, 10 female Wistar rats aged 6months were randomized into 2 groups: Control Group (C): non-ovariectomized rats; Ovariectomy Group (OV): ovariectomized rats to induce osteoporosis. The fluorescence spectroscopy of the teeth was performed for long-term (until 180days). For ex vivo study, the tooth and femur bone of the Wistar rats were removed at 180days to perform fluorescence spectroscopy using excitation laser at 408 and 532nm and μ-EDXRF for calcium (Ca) and phosphorus (P) analysis. There were no intergroup differences in fluorescence spectra with laser at 408nm (p≥0.05), but there were changes in the fluorescence spectra using laser at 532nm which led to both the wavelength shift and changes in the band area (p<0.05). The concentrations of P and Ca for the dentine and cortical bone, respectively, were significantly reduced in OV (p<0.05). Demineralization leading to loss of tissue quality may be assessed by fluorescence spectroscopy using 532nm laser. These findings corroborate those obtained by μ-EDXRF.

  8. A simple preparation of Ag@graphene nanocomposites for surface-enhanced Raman spectroscopy of fluorescent anticancer drug

    NASA Astrophysics Data System (ADS)

    Meng, Ying; Yan, Xueying; Wang, Yi

    2016-05-01

    A simple method was developed to synthesize Ag@graphene nanocomposites with rough Ag nanoparticles (AgNPs) conjugated with graphene nanosheets, and the nanocomposites could be used as substrates for effective surface-enhanced Raman spectroscopy (SERS) of fluorescent anticancer drug (Dox) since they could not only enhance the Raman signals but also suppress the fluorescent signals.

  9. A reusable sensor for the label-free detection of specific oligonucleotides by surface plasmon fluorescence spectroscopy.

    PubMed

    Nöll, Gilbert; Su, Qiang; Heidel, Björn; Yu, Yaming

    2014-01-01

    The development of a reusable molecular beacon (MB)-based sensor for the label-free detection of specific oligonucleotides using surface plasmon fluorescence spectroscopy (SPFS) as the readout method is described. The MBs are chemisorbed at planar gold surfaces serving as fluorescence quenching units. Target oligonucleotides of 24 bases can be detected within a few minutes at high single-mismatch discrimination rates.

  10. Fluorescence spectroscopy for the detection of potentially malignant disorders of the oral cavity: analysis of 30 cases

    NASA Astrophysics Data System (ADS)

    Francisco, A. L. N.; Correr, W. R.; Azevedo, L. H.; Galletta, V. K.; Pinto, C. A. L.; Kowalski, L. P.; Kurachi, C.

    2014-01-01

    Oral cancer is a major health problem worldwide and although early diagnosis of potentially malignant and malignant diseases is associated with better treatment results, a large number of cancers are initially misdiagnosed, with unfortunate consequences for long-term survival. Fluorescence spectroscopy is a noninvasive modality of diagnostic approach using induced fluorescence emission in tumors that can improve diagnostic accuracy. The objective of this study was to determine the ability to discriminate between normal oral mucosa and potentially malignant disorders by fluorescence spectroscopy. Fluorescence investigation under 408 and 532 nm excitation wavelengths was performed on 60 subjects, 30 with potentially malignant disorders and 30 volunteers with normal mucosa. Data was analyzed to correlate fluorescence patterns with clinical and histopathological diagnostics. Fluorescence spectroscopy used as a point measurement technique resulted in a great variety of spectral information. In a qualitative analysis of the fluorescence spectral characteristics of each type of injury evaluated, it was possible to discriminate between normal and abnormal oral mucosa. The results show the potential use of fluorescence spectroscopy for an improved discrimination of oral disorders.

  11. Conformation states of gramicidin A along the pathway to the formation of channels in model membranes determined by 2D NMR and circular dichroism spectroscopy.

    PubMed

    Abdul-Manan, N; Hinton, J F

    1994-06-07

    Gramicidin A incorporated into SDS (sodium dodecyl sulfate) micelles exists as a right-handed, N-to-N-terminal beta 6.3 helical dimer [Lomize, A. L., Orechov, V. Yu., & Arseniev, A.S. (1992) Bioorg. Khim. 18, 182-189]. In the incorporation procedure to achieve the ion channel state of gramicidin A in SDS micelles, trifluoroethanol (TFE) is used to solubilize the hydrophobic peptide before addition to the aqueous/micelle solution. The conformational transition of gramicidin A to form ion channels in SDS micelles, i.e., in TFE and 10% TFE/water, has been investigated using 2D NMR and CD spectroscopy. In neat TFE, gramicidin A was found to be monomeric and may possibly exist in an equilibrium of rapidly interconverting conformers of at least three different forms believed to be left- and/or right-handed alpha and beta 4.4 helices. It was found that the interconversion between these conformers was slowed down in 55% TFE as evident by the observation of at least three different sets of d alpha N COSY peaks although CD gave a net spectrum similar to that in neat TFE. In 10% TFE gramicidin A spontaneously forms a precipitate. The precipitated species were isolated and solubilized in dioxane where gramicidin conformers undergo very slow interconversion and could be characterized by NMR. At least seven different gramicidin A conformations were found in 10% TFE. Four of thes are the same types of double helices as previously found in ethanol (i.e., a symmetric left-handed parallel beta 5.6 double helix, an unsymmetric left-handed parallel beta 5.6 double helix, a symmetric left-handed antiparallel beta 5.6 double helix, a symmetric right-handed parallel beta 5.6 double helix); the fifth is possibly a symmetric right-handed antiparallel beta 5.6 double helix. There is also evidence for the presence of at least one form of monomeric species. Previous observation on the solvent history dependence in the ease of channel incorporation may be explained by the presence of several

  12. Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways.

    PubMed

    Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

    2015-05-01

    We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

  13. Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways

    NASA Astrophysics Data System (ADS)

    Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

    2015-05-01

    We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

  14. Molar concentration from sequential 2-D water-window X-ray ptychography and X-ray fluorescence in hydrated cells

    PubMed Central

    Jones, M. W. M.; Elgass, K. D.; Junker, M. D.; de Jonge, M. D.; van Riessen, G. A.

    2016-01-01

    Recent developments in biological X-ray microscopy have allowed structural information and elemental distribution to be simultaneously obtained by combining X-ray ptychography and X-ray fluorescence microscopy. Experimentally, these methods can be performed simultaneously; however, the optimal conditions for each measurement may not be compatible. Here, we combine two distinct measurements of ultrastructure and elemental distribution, with each measurement performed under optimised conditions. By combining optimised ptychography and fluorescence information we are able to determine molar concentrations from two-dimensional images, allowing an investigation into the interactions between the environment sensing filopodia in fibroblasts and extracellular calcium. Furthermore, the biological ptychography results we present illustrate a point of maturity where the technique can be applied to solve significant problems in structural biology. PMID:27067957

  15. Fluorescence spectroscopy of soil pellets : The use of CP/PARAFAC.

    NASA Astrophysics Data System (ADS)

    Mounier, Stéphane; Nicolodeli, Gustavo; Redon, Roland; Hacherouf, Kalhed; Milori, Debora M. B. P.

    2014-05-01

    Fluorescence spectroscopy is one of the most sensitive techniques available for analytical purposes. It is relatively easy to implement, phenomenologically straightforward and well investigated. Largely non-invasive and fast, so that it can be useful for environmental applications. Fluorescence phenomenon is highly probable in molecular systems containing atoms with lone pairs of electrons such as C=O, aromatic, phenolic, quinone and more rigid unsaturated conjugated systems. These functional groups are present in humic substances (HS) from soils (Senesi, 1990; N. Senesi et al., 1991) and represent the main fluorophors of Soil Organic Matter (SOM). The extension of the conjugated electronic system, the level of heteroatom substitution and type and number of substituting groups under the aromatic rings strongly affect the intensity and wavelength of molecular fluorescence. However, to analyse the SOM it is generally done a chemical extraction that allows measuring the fluorescence response of the liquid extract. To avoid this fractionation of the SOM, Milori et al. (2006) proposed the application of laser induced fluorescence spectroscopy (LIFS) in whole soil. This work intends to assess the technical feasibility of 3D fluorescence spectroscopy using lamp for excitation to analyse solids opaque samples prepared with different substances. Seventy four (74) solid samples were prepared from different mixtures of boric acid (BA), humic substance acid and tryptophan (TRP) powder. The compounds were mixture and a pellet was done by using pressure (8 ton). The pellets were measured using a spectrofluorimeter HITACHI F4500, and a 3D fluorescence tensor was done from emission spectra (200-600 nm) with excitation range from 200 to 500 nm. The acquisition parameters were: step at 5 nm, scan speed at 2400 nm.min-1, response time at 0.1 s, excitation and emission slits at 5 nm and photomultiplier voltage at 700 V. Furthermore, measures of Laser-induced Fluorescence were

  16. Lifetime measurements on electronically excited C 2(A 1Π u) and C 2(d 3Π g) by laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    Bauer, W.; Becker, K. H.; Bielefeld, M.; Meuser, R.

    1986-01-01

    The radiative lifetimes of C 2(A 1Π u, ν'= 0 and 3) were determined by laser-induced fluorescence to be τ 0(0) = 18.5 ± 3 and τ 0(3) = 11.4 ± 2 μs. In addition, the lifetimes of C 2C 2(A 1Π u,ν' = 0 and 1) were measured as τ 0 = 106 ± 15 ns and 105 ± 15 ns, respectively. The error is 3σ.

  17. Fluorescence spectroscopy for the detection of tongue carcinoma--validation in an animal model.

    PubMed

    Kurachi, Cristina; Fontana, Carla R; Rosa, Luiz E B; Bagnato, Vanderlei S

    2008-01-01

    The efficacy of fluorescence spectroscopy to detect squamous cell carcinoma is evaluated in an animal model following laser excitation at 442 and 532 nm. Lesions are chemically induced with a topical DMBA application at the left lateral tongue of Golden Syrian hamsters. The animals are investigated every 2 weeks after the 4th week of induction until a total of 26 weeks. The right lateral tongue of each animal is considered as a control site (normal contralateral tissue) and the induced lesions are analyzed as a set of points covering the entire clinically detectable area. Based on fluorescence spectral differences, four indices are determined to discriminate normal and carcinoma tissues, based on intraspectral analysis. The spectral data are also analyzed using a multivariate data analysis and the results are compared with histology as the diagnostic gold standard. The best result achieved is for blue excitation using the KNN (K-nearest neighbor, a interspectral analysis) algorithm with a sensitivity of 95.7% and a specificity of 91.6%. These high indices indicate that fluorescence spectroscopy may constitute a fast noninvasive auxiliary tool for diagnostic of cancer within the oral cavity.

  18. The modified fluorescence based vesicle fluctuation spectroscopy technique for determination of lipid bilayer bending properties.

    PubMed

    Drabik, Dominik; Przybyło, Magda; Chodaczek, Grzegorz; Iglič, Aleš; Langner, Marek

    2016-02-01

    Lipid bilayer is the main constitutive element of biological membrane, which confines intracellular space. The mechanical properties of biological membranes may be characterized by various parameters including membrane stiffness or membrane bending rigidity, which can be measured using flicker noise spectroscopy. The flicker noise spectroscopy exploits the spontaneous thermal undulations of the membrane. The method is based on the quantitative analysis of a series of microscopic images captured during thermal membrane fluctuations. Thus, measured bending rigidity coefficient depends on the image quality as well as the selection of computational tools for image processing and mathematical model used. In this work scanning and spinning disc confocal microscopies were used to visualize fluctuating membranes of giant unilamellar vesicles. The bending rigidity coefficient was calculated for different acquisition modes, using different fluorescent probes and different image processing methods. It was shown that both imaging approaches gave similar bending coefficient values regardless of acquisition time. Using the developed methodology the effect of fluorescent probe type and aqueous phase composition on the value of the membrane bending rigidity coefficient was measured. Specifically it was found that the bending rigidity coefficient of DOPC bilayer in water is smaller than that determined for POPC membrane. It has been found that the POPC and DOPC bending rigidities coefficient in sucrose solution was lower than that in water. Fluorescence imaging makes possible the quantitative analysis of membrane mechanical properties of inhomogeneous membrane.

  19. Tissue classification and diagnostics using a fiber probe for combined Raman and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Cicchi, Riccardo; Anand, Suresh; Crisci, Alfonso; Giordano, Flavio; Rossari, Susanna; De Giorgi, Vincenzo; Maio, Vincenza; Massi, Daniela; Nesi, Gabriella; Buccoliero, Anna Maria; Guerrini, Renzo; Pimpinelli, Nicola; Pavone, Francesco S.

    2015-07-01

    Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnostic purposes on various tissues in a good agreement with common routine histology. This study included skin, brain and bladder tissues and in particular the classification of: malignant melanoma against melanocytic lesions and healthy skin; urothelial carcinoma against healthy bladder mucosa; brain tumor against dysplastic brain tissue. The diagnostic capabilities were determined using a cross-validation method with a leave-one-out approach, finding very high sensitivity and specificity for all the examined tissues. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

  20. Tissue classification and diagnostics using a fiber probe for combined Raman and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Cicchi, Riccardo; Anand, Suresh; Rossari, Susanna; Sturiale, Alessandro; Giordano, Flavio; De Giorgi, Vincenzo; Maio, Vincenza; Massi, Daniela; Nesi, Gabriella; Buccoliero, Anna Maria; Tonelli, Francesco; Guerrini, Renzo; Pimpinelli, Nicola; Pavone, Francesco S.

    2015-03-01

    Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnostic purposes on various tissues in a good agreement with common routine histology. This study included skin, brain and bladder tissues and in particular the classification of: malignant melanoma against melanocytic lesions and healthy skin; urothelial carcinoma against healthy bladder mucosa; brain tumor against dysplastic brain tissue. The diagnostic capabilities were determined using a cross-validation method with a leave-one-out approach, finding very high sensitivity and specificity for all the examined tissues. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

  1. Laser Induced Fluorescence Spectroscopy of Neutral and Ionized Polycyclic Aromatic Hydrocarbons in the Cosmic Simulation Chamber

    NASA Technical Reports Server (NTRS)

    Bejaoui, Salma; Salama, Farid; Contreras, Cesar; Sciamma O'Brien, Ella; Foing, Bernard; Pascale, Ehrenfreund

    2015-01-01

    Polycyclic aromatic hydrocarbon (PAH) molecules are considered the best carriers to account for the ubiquitous infrared emission bands. PAHs have also been proposed as candidates to explain the diffuse interstellar bands (DIBs), a series of absorption features seen on the interstellar extinction curve and are plausible carriers for the extended red emission (ERE), a photoluminescent process associated with a wide variety of interstellar environments. Extensive efforts have been devoted over the past two decades to characterize the physical and chemical properties of PAH molecules and ions in space. Absorption spectra of PAH molecules and ions trapped in solid matrices have been compared to the DIBs. Absorption spectra of several cold, isolated gas-phase PAHs have also been measured under experimental conditions that mimic the interstellar conditions. The purpose of this study is to provide a new dimension to the existing spectroscopic database of neutral and single ionized PAHs that is largely based on absorption spectra by adding emission spectroscopy data. The measurements are based on the laser induced fluorescence (LIF) technique and are performed with the Pulsed Discharge Nozzle (PDN) of the COSmIC laboratory facility at NASA Ames laboratory. The PDN generates a plasma in a free supersonic jet expansion to simulate the physical and the chemical conditions in interstellar environments. We focus, here, on the fluorescence spectra of large neutral PAHs and their cations where there is a lack of fluorescence spectroscopy data. The astronomical implications of the data (e.g., ERE) are examined.

  2. Authentication of the botanical origin of honey by front-face fluorescence spectroscopy. A preliminary study.

    PubMed

    Ruoff, Kaspar; Karoui, Romdhane; Dufour, Eric; Luginbühl, Werner; Bosset, Jacques-Olivier; Bogdanov, Stefan; Amado, Renato

    2005-03-09

    The potential of front-face fluorescence spectroscopy for the authentication of unifloral and polyfloral honey types (n = 57 samples) previously classified using traditional methods such as chemical, pollen, and sensory analysis was evaluated. Emission spectra were recorded between 280 and 480 nm (excit: 250 nm), 305 and 500 nm (excit: 290 nm), and 380 and 600 nm (excit: 373 nm) directly on honey samples. In addition, excitation spectra (290-440 nm) were recorded with the emission measured at 450 nm. A total of four different spectral data sets were considered for data analysis. After normalization of the spectra, chemometric evaluation of the spectral data was carried out using principal component analysis (PCA) and linear discriminant analysis (LDA). The rate of correct classification ranged from 36% to 100% by using single spectral data sets (250, 290, 373, 450 nm) and from 73% to 100% by combining these four data sets. For alpine polyfloral honey and the unifloral varieties investigated (acacia, alpine rose, honeydew, chestnut, and rape), correct classification ranged from 96% to 100%. This preliminary study indicates that front-face fluorescence spectroscopy is a promising technique for the authentication of the botanical origin of honey. It is nondestructive, rapid, easy to use, and inexpensive. The use of additional excitation wavelengths between 320 and 440 nm could increase the correct classification of the less characteristic fluorescent varieties.

  3. Multimodal fiber probe spectroscopy for tissue diagnostics applications: a combined Raman-fluorescence approach

    NASA Astrophysics Data System (ADS)

    Cicchi, Riccardo; Anand, Suresh; Rossari, Susanna; Sturiale, Alessandro; Giordano, Flavio; De Giorgi, Vincenzo; Maio, Vincenza; Massi, Daniela; Nesi, Gabriella; Buccoliero, Anna Maria; Tonelli, Francesco; Guerrini, Renzo; Pimpinelli, Nicola; Pavone, Francesco S.

    2014-03-01

    Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnosing melanocytic lesions in a good agreement with common routine histology. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. Further investigations were performed on colon and brain tissue samples in order to have a benchmark for diagnosing a broader range of tissue lesions and malignancies. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

  4. Surface plasmon field-enhanced fluorescence spectroscopy studies of primer extension reactions.

    PubMed

    Stengel, Gudrun; Knoll, Wolfgang

    2005-04-22

    Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) utilizes the evanescent electromagnetic field of a surface plasmon to excite chromophors in close proximity to the surface. While conventional surface plasmon resonance spectroscopy allows the observation of surface reactions by means of refractive index changes, SPFS additionally provides a channel for the read-out of fluorescence changes. Thus, the detection limit for low mass compounds, whose adsorption is only accompanied by small refractive index changes, can be substantially improved by fluorescent labeling. In this study, we present the first example that utilizes SPFS to follow the dynamics of an enzymatic reaction. The elongation of surface-tethered DNA has been observed by the incorporation of Cy5-labeled nucleotides into the nascent strand by the action of DNA polymerase I (Klenow fragment). The technique offers a rapid way to determine the binding constant and the catalytic activity of a DNA processing enzyme, here exemplified by the Klenow fragment. Furthermore, the effect of mispaired bases in the primer/template duplex and the influence of different label densities have been studied. The resulting sensitivity for nucleotide incorporation, being in the femtomolar regime, combined with the specificity of the enzyme for fully complementary DNA duplexes suggest the application of this assay as a powerful tool for DNA detection.

  5. Biosensor platform based on surface plasmon-enhanced fluorescence spectroscopy and responsive hydrogel binding matrix

    NASA Astrophysics Data System (ADS)

    Huang, Chun-Jen; Jonas, Ulrich; Dostálek, Jakub; Knoll, Wolfgang

    2009-05-01

    We report a novel biosensor platform based on surface plasmon-enhanced fluorescence spectroscopy (SPFS) and a responsive N-isopropylacrylamide (NIPAAm) hydrogel binding matrix. This binding matrix highly swells in aqueous environment and it can be modified with receptor biomolecules by using active ester coupling chemistry. After the binding of target analyte molecules contained in a sample by receptor biomolecules immobilized in the hydrogel matrix, the captured analyte molecules can be compacted on the surface through the collapse of the gel triggered by an external stimulus. A thin hydrogel NIPAAm-based film was attached to a gold sensor surface and modified with mouse IgG receptor molecules. The affinity binding of antibodies against mouse IgG that were labeled with Alexa Fluor chromophores was observed by surface plasmon-enhanced fluorescence spectroscopy. We demonstrate that the collapse of the hydrogel matrix results in the enhancement of measured fluorescence intensity owing to the increase in the concentration of captured molecules within the evanescent field of surface plasmons.

  6. Experimental and clinical evaluation of a spectroscopy system for fluorescence-guided excimer laser angioplasty

    NASA Astrophysics Data System (ADS)

    Morguet, Andreas J.; Gabriel, Ruth E.; Buchwald, Arnd B.

    1996-12-01

    This study evaluated a single-laser approach for simultaneous ablation and fluorescence excitation for spectroscopic guidance of laser angioplasty. A spectroscopy system was developed and coupled to a clinical XeCl excimer laser. Ablation of 162 human aortic samples in saline and blood with 45 mJ/mm2 per pulse yielded 676 fluorescence spectra validated histologically. Five types of spectra could be differentiated: atheroma, fibrous plaque, calcified lesion in saline, normal media and calcified lesion in blood. Discriminant analysis prospectively classified 576 validation spectra with a sensitivity between 83.5 and 100 percent and a specificity between 96.8 and 100 percent. Subsequently, the equipment was used in 16 patients for angioplasty of 18 coronary stenoses applying 500 to 1725 pulses with 45 to 60 mJ/mm2 under saline flushing. A total of 783 spectra were recorded and validated by intracoronary ultrasound. Except for the media spectrum, all types of spectra were observed in vivo, too. The predominant sonographic category also prevailed in spectroscopy. In conclusion, using an excimer laser for angioplasty allows combining ablation and fluorescence excitation without a diagnostic laser. Principal types of atherosclerotic lesions and the media can be differentiated spectroscopically with this approach.

  7. Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy.

    PubMed

    Korlann, You; Dertinger, Thomas; Michalet, Xavier; Weiss, Shimon; Enderlein, Jörg

    2008-09-15

    We present a new technique, polarization-modulation dual-focus fluorescence correlation spectroscopy (pmFCS), based on the recently intro-duced dual-focus fluorescence correlation spectroscopy (2fFCS) to measure the absolute value of diffusion coefficients of fluorescent molecules at pico- to nanomolar concentrations. Analogous to 2fFCS, the new technique is robust against optical saturation in yielding correct values of the diffusion coefficient. This is in stark contrast to conventional FCS where optical saturation leads to an apparent decrease in the determined diffusion coefficient with increasing excitation power. However, compared to 2fFCS, the new technique is simpler to implement into a conventional confocal microscope setup and is compatible with cw-excitation, only needing as add-ons an electro-optical modulator and a differential interference contrast prism. With pmFCS, the measured diffusion coefficient (D) for Atto655 maleimide in water at 25?C is determined to be equal to (4.09 +/- 0.07) x 10(-6)cm(2)/s, in good agreement with the value of 4.04 x 10-6cm2/s as measured by 2fFCS.

  8. High pressure sample cell for total internal reflection fluorescence spectroscopy at pressures up to 2500 bar

    NASA Astrophysics Data System (ADS)

    Koo, Juny; Czeslik, Claus

    2012-08-01

    Total internal reflection fluorescence (TIRF) spectroscopy is a surface sensitive technique that is widely used to characterize the structure and dynamics of molecules at planar liquid-solid interfaces. In particular, biomolecular systems, such as protein adsorbates and lipid membranes can easily be studied by TIRF spectroscopy. Applying pressure to molecular systems offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. So far, most of these volume changes have been characterized in bulk solution, only. Here, we describe the design and performance of a high pressure sample cell that allows for TIRF spectroscopy under high pressures up to 2500 bar (2.5 × 108 Pa), in order to expand the understanding of volume effects from the bulk phase to liquid-solid interfaces. The new sample cell is based on a cylindrical body made of Nimonic 90 alloy and incorporates a pressure transmitting sample cuvette. This cuvette is composed of a fused silica prism and a flexible rubber gasket. It contains the sample solution and ensures a complete separation of the sample from the liquid pressure medium. The sample solution is in contact with the inner wall of the prism forming the interface under study, where fluorescent molecules are immobilized. In this way, the new high pressure TIRF sample cell is very useful for studying any biomolecular layer that can be deposited at a planar water-silica interface. As examples, high pressure TIRF data of adsorbed lysozyme and two phospholipid membranes are presented.

  9. [Identifying the origin of chromophoric dissolved organic matter in Xiamen Bay using fluorescence spectroscopy and chemometrics].

    PubMed

    Lin, Hui; Guo, Wei-Dong; Xu, Jing; Hu, Ming-Hui

    2013-02-01

    The fluorescent components of chromophoric dissolved matter (CDOM) in water samples collected from Xiamen Bay in spring and autumn, 2009 were examined using excitation-emission matrix fluorescence spectroscopy combined with parallel factor analysis (EEMs-PARAFAC). PARAFAC decomposed the fluorescence matrices of CDOM into three humic-like (C1: 250, 345/454 nm; C2: 230, 310/374 nm; C5: 265, 424/478 nm) and two protein-like (C3: 230/342 nm; C4: 230, 275/322 nm) components. Good linear correlation occurred among three humic-like components and between two protein-like components, respectively. This demonstrated that the same types of components (humic-like or protein-like) have similar origin and geochemical behaviors. High abundances of humic-like components were found at the upstream zone of the Jiulong Estuary, while the high abundance of protein-like components occurred at the northern part of semi-enclosed Western Xiamen Harbor. The significant negative correlations were found between the abundances of all fluorescence components and salinity in the estuary area. However, the high contents of chlorophyll a were in line with the high abundances of C3 and C4 in non-estuarine area, which implies that phytoplankton activity could be another important source of protein-like components besides the river runoff. A principal component analysis(PCA) of fluorescent components revealed that terrestrial runoff was the dominant sources of CDOM fluorescence components in Xiamen Bay, while the contribution of the in situ biological processes was relatively lower. This study demonstrates that the combination use of PARAFAC modeling and chemometrics (i. e. PCA) is very useful in identifying the origin of CDOM and quantifying the primary factors influencing their distributions.

  10. Histologic differences between orthotopic xenograft pancreas models affect Verteporfin uptake measured by fluorescence microscopy and spectroscopy

    NASA Astrophysics Data System (ADS)

    O'Hara, Julia A.; Samkoe, Kimberley S.; Chen, Alina; Isabelle, Martin; Hoopes, P. J.; Hasan, Tayyaba; Pogue, Brian W.

    2012-02-01

    Photodynamic therapy (PDT) that uses the second generation photosensitizer, verteporfin (VP), is a developing therapy for pancreatic cancer. The optimal timing of light delivery related to VP uptake and distribution in pancreatic tumors will be important information to obtain to improve treatment for this intractable disease. In this work we examined uptake and distribution of VP in two orthotopic pancreatic tumors with different histological structure. ASPC-1 (fast-growing) and Panc-1 (slower growing) tumors were implanted in SCID mice and studied when tumors were approximately 100mm3. In a pilot study, these tumors had been shown to differ in uptake of VP using lightinduced fluorescence spectroscopy (LIFS) in vivo and fluorescence imaging ex vivo and that work is extended here. In vivo fluorescence mean readings of tumor and liver increased rapidly up to 15 minutes after photosensitizer injection for both tumor types, and then continued to increase up to 60 minutes post injection to a higher level in ASPC-1 than in Panc-1. There was variability among animals with the same tumor type, in both liver and tumor uptake and no selectivity of tumor over liver. In this work we further examined VP uptake at multiple time points in relation to microvascular density and perfusion, using DiOC7 (to mark blood vessels) and VP fluorescence in the same tissue slices. Analysis of DiOC7 fluorescence indicates that AsPC-1 and Panc-1 have different vascular densities but AsPC-1 vasculature is more perfusive. Analysis of colocalized DiOC7 and VP fluorescence showed ASPC-1 with higher accumulation of VP 3 hrs after injection and more VP at a distance from blood vessels compared to Panc-1. This work shows the need for techniques to analyze photosensitizer distribution in order to optimize photodynamic therapy as an effective treatment for pancreatic tumors.

  11. Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects

    NASA Astrophysics Data System (ADS)

    van den Berg, Petra A. W.; Widengren, Jerker; Hink, Mark A.; Rigler, Rudolf; Visser, Antonie J. W. G.

    2001-09-01

    Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity (<10 kW/cm 2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations (> 50 mM of I -) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the

  12. Substrate-Supported Phospholipid Membranes Studied by Surface Plasmon Resonance and Surface Plasmon Fluorescence Spectroscopy

    PubMed Central

    Tawa, Keiko; Morigaki, Kenichi

    2005-01-01

    Substrate-supported planar lipid bilayer membranes are attractive model cellular membranes for biotechnological applications such as biochips and sensors. However, reliable fabrication of the lipid membranes on solid surfaces still poses significant technological challenges. In this study, simultaneous surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) measurements were applied to the monitoring of adsorption and subsequent reorganization of phospholipid vesicles on solid substrates. The fluorescence intensity of SPFS depends very sensitively on the distance between the gold substrate and the fluorophore because of the excitation energy transfer to gold. By utilizing this distance dependency, we could obtain information about the topography of the adsorbed membranes: Adsorbed vesicles could be clearly distinguished from planar bilayers due to the high fluorescence intensity. SPSF can also incorporate various analytical techniques to evaluate the physicochemical properties of the adsorbed membranes. As an example, we demonstrated that the lateral mobility of lipid molecules could be estimated by observing the recovery of fluorescence after photobleaching. Combined with the film thickness information obtained by SPR, SPR-SPFS proved to be a highly informative technique to monitor the lipid membrane assembly processes on solid substrates. PMID:16040759

  13. Fluorescence and Absorbance Spectroscopy Methods to Study Membrane Perturbations by Antimicrobial Host Defense Peptides.

    PubMed

    Arias, Mauricio; Vogel, Hans J

    2017-01-01

    Antimicrobial peptides (AMPs) are currently intensely studied because of their potential as new bactericidal and bacteriostatic agents. The mechanism of action of numerous AMPs involves the permeabilization of bacterial membranes. Several methods have been developed to study peptide-membrane interactions; in particular optical spectroscopy methods are widely used. The intrinsic fluorescence properties of the Trp indole ring in Trp-containing AMPs can be exploited by measuring the fluorescence blue shift and acrylamide-induced fluorescence quenching. One important aspect of such studies is the use of distinct models of the bacterial membrane, in most cases large unilamellar vesicles (LUVs) with different, yet well-defined, phospholipid compositions. Deploying LUVs that are preloaded with fluorescent dyes, such as calcein, also allows for the study of vesicle permeabilization by AMPs. In addition, experiments using genetically engineered live Escherichia coli cells can be used to distinguish between the effects of AMPs on the outer and inner membranes of gram-negative bacteria. In combination, these methods can provide a detailed insight into the mode of action of AMPs.

  14. Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

    PubMed

    Laňková, Martina; Humpolíčková, Jana; Vosolsobě, Stanislav; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan

    2016-04-01

    A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

  15. Fluorescence spectroscopy for tongue carcinoma detection: study in an animal model

    NASA Astrophysics Data System (ADS)

    Kurachi, Cristina; Fontana, Carla R.; Melo, Claudia A. S.; Rosa, Luiz E. B.; Bagnato, Vanderlei S.

    2005-04-01

    Fluorescence diagnosis of malignant lesions has been showed as an attractive optical technique due especially to its real-time response and a more objective and quantitative evaluation. Even though the oral cavity allows a direct examination many lesions are diagnosed when it is already in advanced stage, compromising the patient prognosis. In this study, the fluorescence spectroscopy was used to the detection of chemically induced carcinoma at the lateral border of the tongue in a hamster model. Two excitations wavelengths in visible region were applied: 442 and 532 nm. All the spectra results were analyzed comparing with the histopathological diagnosis. The better results were achieved with the 442 nm laser excitation. The spectra from carcinoma showed new emission bands and these were used to determined different ratios for a quantitative analysis. Using the 625-645 nm fluorescence range under 442 nm excitation (A3 coefficient) the percentage of false negative was of 9.1%, however the false positive percentage was of 18.5%. The 532 nm excitation provided a better normal tissue detection compared to 442 nm excitation. The ideal clinical condition is probably the use of multiple wavelengths excitation for a broader tissue fluorescence investigation.

  16. Fluorescence spectroscopy of collagen crosslinking: non-invasive and in situ evaluation of corneal stiffness

    NASA Astrophysics Data System (ADS)

    Franco, Walfre; Ortega-Martinez, Antonio; Zhu, Hong; Wang, Ruisheng; Kochevar, Irene E.

    2015-03-01

    Collagen is a long fibrous structural protein that imparts mechanical support, strength and elasticity to many tissues. The state of the tissue mechanical environment is related to tissue physiology, disease and function. In the cornea, the collagen network is responsible for its shape and clarity; disruption of this network results in degradation of visual acuity, for example in the keratoconus eye disease. The objective of the present study is to investigate the feasibility of using the endogenous fluorescence of collagen crosslinks to evaluate variations in the mechanical state of tissue, in particular, the stiffness of cornea in response to different degrees of photo-crosslinking or RGX treatment—a novel keratoconus treatment. After removing the epithelium, rabbit corneas were stained with Rose Bengal and then irradiated with a 532 nm solid-state laser. Analysis of the excitation spectra obtained by fluorescence spectroscopy shows a correlation between the fluorescence intensity at 370/460 nm excitation/emission wavelengths and the mechanical properties. In principle, it may be feasible to use the endogenous fluorescence of collagen crosslinks to evaluate the mechanical stiffness of cornea non-invasively and in situ.

  17. Applications of time-resolved laser fluorescence spectroscopy to the environmental biogeochemistry of actinides.

    PubMed

    Collins, Richard N; Saito, Takumi; Aoyagi, Noboru; Payne, Timothy E; Kimura, Takaumi; Waite, T David

    2011-01-01

    Time-resolved laser fluorescence spectroscopy (TRLFS) is a useful means of identifying certain actinide species resulting from various biogeochemical processes. In general, TRLFS differentiates chemical species of a fluorescent metal ion through analysis of different excitation and emission spectra and decay lifetimes. Although this spectroscopic technique has largely been applied to the analysis of actinide and lanthanide ions having fluorescence decay lifetimes on the order of microseconds, such as UO , Cm, and Eu, continuing development of ultra-fast and cryogenic TRLFS systems offers the possibility to obtain speciation information on metal ions having room-temperature fluorescence decay lifetimes on the order of nanoseconds to picoseconds. The main advantage of TRLFS over other advanced spectroscopic techniques is the ability to determine in situ metal speciation at environmentally relevant micromolar to picomolar concentrations. In the context of environmental biogeochemistry, TRLFS has principally been applied to studies of (i) metal speciation in aqueous and solid phases and (ii) the coordination environment of metal ions sorbed to mineral and bacterial surfaces. In this review, the principles of TRLFS are described, and the literature reporting the application of this methodology to the speciation of actinides in systems of biogeochemical interest is assessed. Significant developments in TRLFS methodology and advanced data analysis are highlighted, and we outline how these developments have the potential to further our mechanistic understanding of actinide biogeochemistry.

  18. Substrate-supported phospholipid membranes studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy.

    PubMed

    Tawa, Keiko; Morigaki, Kenichi

    2005-10-01

    Substrate-supported planar lipid bilayer membranes are attractive model cellular membranes for biotechnological applications such as biochips and sensors. However, reliable fabrication of the lipid membranes on solid surfaces still poses significant technological challenges. In this study, simultaneous surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) measurements were applied to the monitoring of adsorption and subsequent reorganization of phospholipid vesicles on solid substrates. The fluorescence intensity of SPFS depends very sensitively on the distance between the gold substrate and the fluorophore because of the excitation energy transfer to gold. By utilizing this distance dependency, we could obtain information about the topography of the adsorbed membranes: Adsorbed vesicles could be clearly distinguished from planar bilayers due to the high fluorescence intensity. SPSF can also incorporate various analytical techniques to evaluate the physicochemical properties of the adsorbed membranes. As an example, we demonstrated that the lateral mobility of lipid molecules could be estimated by observing the recovery of fluorescence after photobleaching. Combined with the film thickness information obtained by SPR, SPR-SPFS proved to be a highly informative technique to monitor the lipid membrane assembly processes on solid substrates.

  19. What information is contained in the fluorescence correlation spectroscopy curves, and where

    NASA Astrophysics Data System (ADS)

    Khadem, S. M. J.; Hille, C.; Löhmannsröben, H.-G.; Sokolov, I. M.

    2016-08-01

    We discuss the application of fluorescence correlation spectroscopy (FCS) for characterization of anomalous diffusion of tracer particles in crowded environments. While the fact of anomaly may be detected by the standard fitting procedure, the value of the exponent α of anomalous diffusion may be not reproduced correctly for non-Gaussian anomalous diffusion processes. The important information is however contained in the asymptotic behavior of the fluorescence autocorrelation function at long and at short times. Thus, analysis of the short-time behavior gives reliable values of α and of lower moments of the distribution of particles' displacement, which allows us to confirm or reject its Gaussian nature. The method proposed was tested on the FCS data obtained in artificial crowded fluids and in living cells.

  20. Comparing Raman and fluorescence lifetime spectroscopy from human atherosclerotic lesions using a bimodal probe.

    PubMed

    Dochow, Sebastian; Fatakdawala, Hussain; Phipps, Jennifer E; Ma, Dinglong; Bocklitz, Thomas; Schmitt, Michael; Bishop, John W; Margulies, Kenneth B; Marcu, Laura; Popp, Jürgen

    2016-09-01

    Fluorescence lifetime imaging (FLIm) and Raman spectroscopy are two promising methods to support morphological intravascular imaging techniques with chemical contrast. Both approaches are complementary and may also be used in combination with OCT/IVUS to add chemical specificity to these morphologic intravascular imaging modalities. In this contribution, both modalities were simultaneously acquired from two human coronary specimens using a bimodal probe. A previously trained SVM model was used to interpret the fluorescence lifetime data; integrated band intensities displayed in RGB false color images were used to interpret the Raman data. Both modalities demonstrate unique strengths and weaknesses and these will be discussed in comparison to histologic analyses from the two coronary arteries imaged.

  1. Measurement of the temperature-dependent diffusion properties of nanoparticles by using fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Jung, Chanbae; Lee, Jaeran; Kang, Manil; Kim, Sok Won

    2014-10-01

    Changes in the diffusion properties of three kinds of fluorescent particles, Alexa Fluor 647, Q-dots (quantum dots), and beads, with temperature were investigated with a home-built fluorescence correlation spectroscopy (FCS) system based on a confocal microscope. In all samples, as the temperature was increased, the diffusion times were reduced, indicating an increase in the diffusion coefficient. In particular, of all the particles, Alexa Fluor 647 having the smallest size of ˜1 nm, showed a hydrodynamic radius that increased with increasing temperature of the solvent. However, for the Q-dots and beads with larger sizes, the hydrodynamic radius of the particles was inversely proportional to the temperature. These results show that diffusion coefficient obtained by changing the temperature has an influence on the hydrodynamic radius of the particles.

  2. Application of fluorescence spectroscopy for on-line bioprocess monitoring and control

    NASA Astrophysics Data System (ADS)

    Boehl, Daniela; Solle, D.; Toussaint, Hans J.; Menge, M.; Renemann, G.; Lindemann, Carsten; Hitzmann, Bernd; Scheper, Thomas-Helmut

    2001-02-01

    12 Modern bioprocess control requires fast data acquisition and in-time evaluation of bioprocess variables. On-line fluorescence spectroscopy for data acquisition and the use of chemometric methods accomplish these requirements. The presented investigations were performed with fluorescence spectrometers with wide ranges of excitation and emission wavelength. By detection of several biogenic fluorophors (amino acids, coenzymes and vitamins) a large amount of information about the state of the bioprocess are obtained. For the evaluation of the process variables partial least squares regression is used. This technique was applied to several bioprocesses: the production of ergotamine by Claviceps purpurea, the production of t-PA (tissue plasminogen activator) by animal cells and brewing processes. The main point of monitoring the brewing processes was to determine the process variables cell count and extract concentration.

  3. Fluorescence Spectroscopy of the Retina for the Screening of Bovine Spongiform Encephalopathy.

    PubMed

    Bhattacharjee, Ujjal; Graham, Catherine; Czub, Stefanie; Dudas, Sandor; Rasmussen, Mark A; Casey, Thomas A; Petrich, Jacob W

    2016-01-13

    Transmissible spongiform encephalopathies (TSE) are progressive, neurodegenerative disorders, of which bovine spongiform encephalopathy (BSE) is of special concern because it is infectious and debilitating to humans. The possibility of using fluorescence spectroscopy to screen for BSE in cattle was explored. Fluorescence spectra from the retinas of experimentally infected BSE-positive cattle with clinical disease were compared with those from both sham-inoculated and non-inoculated BSE-negative cattle. The distinct intensity difference of about 4-10-fold between the spectra of the BSE-positive and the BSE-negative (sham-inoculated and non-inoculated) eyes suggests the basis for a means of developing a rapid, noninvasive examination of BSE in particular and TSEs in general.

  4. Fluorescence spectroscopy to study dissolved organic matter interactions with agrochemicals applied in Swiss vineyards.

    PubMed

    Daouk, Silwan; Frege, Carla; Blanc, Nicolas; Mounier, Stéphane; Redon, Roland; Merdy, Patricia; Lucas, Yves; Pfeifer, Hans-Rudolf

    2015-06-01

    UV/Vis fluorescence spectroscopy was used to study the possible interactions of dissolved organic matter (DOM) with the herbicide glyphosate and copper-based fungicide used in vineyards. The study focused on the role of DOM in the transport of these micropollutants from parcels to surface waters (river, lake). Soil solution and river water samples were collected in the Lavaux vineyard area, western Switzerland. Their fluorescence excitation emission matrices (EEM) were decomposed using parallel factor (PARAFAC) analysis, and compared to their content in glyphosate and copper. PARAFAC analysis of EEM of both types of samples showed the contribution of protein-like and humic-like fluorophores. In soil water samples, complexes between fulvic-like and humic-like fluorophores of DOM, copper, and glyphosate were likely formed. In surface water, DOM-copper and glyphosate-copper interactions were observed, but not between glyphosate and DOM.

  5. Artificial neural networks for processing fluorescence spectroscopy data in skin cancer diagnostics

    NASA Astrophysics Data System (ADS)

    Lenhardt, L.; Zeković, I.; Dramićanin, T.; Dramićanin, M. D.

    2013-11-01

    Over the years various optical spectroscopic techniques have been widely used as diagnostic tools in the discrimination of many types of malignant diseases. Recently, synchronous fluorescent spectroscopy (SFS) coupled with chemometrics has been applied in cancer diagnostics. The SFS method involves simultaneous scanning of both emission and excitation wavelengths while keeping the interval of wavelengths (constant-wavelength mode) or frequencies (constant-energy mode) between them constant. This method is fast, relatively inexpensive, sensitive and non-invasive. Total synchronous fluorescence spectra of normal skin, nevus and melanoma samples were used as input for training of artificial neural networks. Two different types of artificial neural networks were trained, the self-organizing map and the feed-forward neural network. Histopathology results of investigated skin samples were used as the gold standard for network output. Based on the obtained classification success rate of neural networks, we concluded that both networks provided high sensitivity with classification errors between 2 and 4%.

  6. Vesicle fusion studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy.

    PubMed

    Morigaki, Kenichi; Tawa, Keiko

    2006-08-15

    Substrate-supported planar lipid bilayers are generated most commonly by the adsorption and transformation of phospholipid vesicles (vesicle fusion). We have recently demonstrated that simultaneous measurements of surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) are highly informative for monitoring lipid membranes on solid substrates. SPR and SPFS provide information on the amount and topography of adsorbed lipid membranes, respectively. In this study, the vesicle fusion process was studied in detail by measuring SPR-SPFS at a higher rate and plotting the obtained fluorescence intensity versus film thickness. We could track the initial adsorption of vesicles, the onset of vesicle rupture occurring at certain vesicle coverage of the surface, and the autocatalytic transformation into planar bilayers. We also monitored vesicle fusion of the same vesicle suspensions by quartz crystal microbalance with dissipation monitoring (QCM-D). We compared the results obtained from SPR-SPFS and QCM-D to highlight the unique information provided by SPR-SPFS.

  7. [Measurement and analysis of lead in soil using X-ray fluorescence spectroscopy].

    PubMed

    Zhang, Rong; Zhang, Yu-Jun; Zhang, Wei; Chen, Dong; Yu, Xiao-Ya; Gao, Yan-Wei

    2013-02-01

    The present paper analyzed the characteristics of X-ray fluorescence spectroscopy (XRF) of metal element lead in soil using the NITON XLt793 portable X-ray fluorescence spectra of heavy metal analyzer under laboratory conditions. The characteristic spectral lines of L(alpha) (energy: 10. 55 keV) and L(beta) (energy: 12. 61 keV) with different matrix elements were selected respectively for lead in the experiment. By measuring the intensities of the characteristic spectral line with different Pb concentration, the results demonstrate that the relation between concentration [mass fraction 10 x 10(-6) - 1 800 x 10(-6)] of Pb element and the intensity of the characteristic spectrum is well linear. The calibration curve of Pb was plotted based on the different concentration measurement results, and the limit of detection of 7.89 x 10(-6) was obtained for Pb in soil.

  8. Investigation of polymer electrolyte membrane chemical degradation and degradation mitigation using in situ fluorescence spectroscopy

    PubMed Central

    Prabhakaran, Venkateshkumar; Arges, Christopher G.; Ramani, Vijay

    2012-01-01

    A fluorescent molecular probe, 6-carboxy fluorescein, was used in conjunction with in situ fluorescence spectroscopy to facilitate real-time monitoring of degradation inducing reactive oxygen species within the polymer electrolyte membrane (PEM) of an operating PEM fuel cell. The key requirements of suitable molecular probes for in situ monitoring of ROS are presented. The utility of using free radical scavengers such as CeO2 nanoparticles to mitigate reactive oxygen species induced PEM degradation was demonstrated. The addition of CeO2 to uncatalyzed membranes resulted in close to 100% capture of ROS generated in situ within the PEM for a period of about 7 h and the incorporation of CeO2 into the catalyzed membrane provided an eightfold reduction in ROS generation rate. PMID:22219367

  9. Quantifying the flow dynamics of supercritical CO2-water displacement in a 2D porous micromodel using fluorescent microscopy and microscopic PIV

    NASA Astrophysics Data System (ADS)

    Kazemifar, Farzan; Blois, Gianluca; Kyritsis, Dimitrios C.; Christensen, Kenneth T.

    2016-09-01

    The multi-phase flow of liquid/supercritical CO2 and water (non-wetting and wetting phases, respectively) in a two-dimensional silicon micromodel was investigated at reservoir conditions (80 bar, 24 °C and 40 °C). The fluorescent microscopy and microscopic particle image velocimetry (micro-PIV) techniques were combined to quantify the flow dynamics associated with displacement of water by CO2 (drainage) in the porous matrix. To this end, water was seeded with fluorescent tracer particles, CO2 was tagged with a fluorescent dye and each phase was imaged independently using spectral separation in conjunction with microscopic imaging. This approach allowed simultaneous measurement of the spatially-resolved instantaneous velocity field in the water and quantification of the spatial configuration of the two fluid phases. The results, acquired with sufficient time resolution to follow the dynamic progression of both phases, provide a comprehensive picture of the flow physics during the migration of the CO2 front, the temporal evolution of individual menisci, and the growth of fingers within the porous microstructure. During that growth process, velocity jumps 20-25 times larger in magnitude than the bulk velocity were measured in the water phase and these bursts of water flow occurred both in-line with and against the bulk flow direction. These unsteady velocity events support the notion of pressure bursts and Haines jumps during pore drainage events as previously reported in the literature [1-3]. After passage of the CO2 front, shear-induced flow was detected in the trapped water ganglia in the form of circulation zones near the CO2-water interfaces as well as in the thin water films wetting the surfaces of the silicon micromodel. To our knowledge, the results presented herein represent the first quantitative spatially and temporally resolved velocity-field measurements at high pressure for water displacement by liquid/supercritical CO2 injection in a porous micromodel.

  10. Fluorescence spectroscopy as a tool for quality assessment of humic substances

    NASA Astrophysics Data System (ADS)

    Boguta, Patrycja

    2016-04-01

    *The studies were partly carried out within the framework of a research project. The project was financed from funds of National Science Center on the base of decision number DEC-2013/11/D/NZ9/02545. Fluorescence spectroscopy belongs to modern, non-destructive, rapid and relatively cheap methods, as well as for many years it was successfully used in studies of organic compounds in the fields of medicine, biology and chemistry. On the other hand, soil organic matter is a group of compounds with a complex spatial structure showing a large number of groups with different kinds of fluorophores. This could suggest the possibility of application of fluorescence spectroscopy in assessing the quality of humic substances as well as in monitoring of their chemical transformations. The aim of study was chemical description of humic and fulvic acids based on fluorescence spectra, as well as an attempt of evaluation of changes occurring under the influence of different pH and during interactions with various concentrations of metal. The humic and fulvic acids were isolated from chemically different soils. The measurements were carried out on Hitachi fluorescence spectrometer in solutions with a concentration of humic acids 40mg dm-3, at pH from 3 to 7, and for the evaluation of the metal impact: with increasing Zn concentrations (0-50mg dm-3). The fluorescence spectra were recorded in the form of synchronous and emission-excitation matrices (EEM). Studies have shown the presence of different groups of fluorophores. Synchronous spectra were characterized by a well-separated bands showing fluorescence in the area of low, medium and high wavelengths, suggesting the presence of structures, both weakly and strongly humified. EEM spectra revealed map of fluorophores within wide ranges of emission and excitation. Fluorophores differed in both position and intensity. The highest intensity was observed for compounds with the lowest humification degree which might be due to high amount

  11. Changes in chlorophyll a fluorescence of glyphosate-tolerant soybean plants induced by glyphosate: in vivo analysis by laser-induced fluorescence spectroscopy.

    PubMed

    Fernandes, Joelson; Falco, William Ferreira; Oliveira, Samuel Leite; Caires, Anderson Rodrigues Lima

    2013-05-01

    A significant increase in the use of the herbicide glyphosate has generated many questions about its residual accumulation in the environment and possible damage to crops. In this study, changes in chlorophyll a (chl-a) fluorescence induced by glyphosate in three varieties of glyphosate-resistant soybean plants were determined with an in vivo analysis based on a portable laser-induced fluorescence system. Strong suppression of chl-a fluorescence was observed for all plants treated with the herbicide. Moreover, the ratio of the emission bands in the red and far-red regions (685 nm/735 nm) indicates that the application of glyphosate led to chlorophyll degradation. The results also indicated that the use of glyphosate, even at concentrations recommended by the manufacturer, suppressed chl-a fluorescence. In summary, this study shows that fluorescence spectroscopy can detect, in vivo, very early changes in the photosynthetic status of transgenic soybeans treated with this herbicide.

  12. Identification of the Excited-State C═C and C═O Modes of trans-β-Apo-8'-carotenal with Transient 2D-IR-EXSY and Femtosecond Stimulated Raman Spectroscopy.

    PubMed

    Di Donato, Mariangela; Ragnoni, Elena; Lapini, Andrea; Kardaś, Tomasz M; Ratajska-Gadomska, Boźena; Foggi, Paolo; Righini, Roberto

    2015-05-07

    Assigning the vibrational modes of molecules in the electronic excited state is often a difficult task. Here we show that combining two nonlinear spectroscopic techniques, transient 2D exchange infrared spectroscopy (T2D-IR-EXSY) and femtosecond stimulated Raman spectroscopy (FSRS), the contribution of the C═C and C═O modes in the excited-state vibrational spectra of trans-β-apo-8'-carotenal can be unambiguously identified. The experimental results reported in this work confirm a previously proposed assignment based on quantum-chemical calculations and further strengthen the role of an excited state with charge-transfer character in the relaxation pathway of carbonyl carotenoids. On a more general ground, our results highlight the potentiality of nonlinear spectroscopic methods based on the combined use of visible and infrared pulses to correlate structural and electronic changes in photoexcited molecules.

  13. A deconvolution extraction method for 2D multi-object fibre spectroscopy based on the regularized least-squares QR-factorization algorithm

    NASA Astrophysics Data System (ADS)

    Yu, Jian; Yin, Qian; Guo, Ping; Luo, A.-li

    2014-09-01

    This paper presents an efficient method for the extraction of astronomical spectra from two-dimensional (2D) multifibre spectrographs based on the regularized least-squares QR-factorization (LSQR) algorithm. We address two issues: we propose a modified Gaussian point spread function (PSF) for modelling the 2D PSF from multi-emission-line gas-discharge lamp images (arc images), and we develop an efficient deconvolution method to extract spectra in real circumstances. The proposed modified 2D Gaussian PSF model can fit various types of 2D PSFs, including different radial distortion angles and ellipticities. We adopt the regularized LSQR algorithm to solve the sparse linear equations constructed from the sparse convolution matrix, which we designate the deconvolution spectrum extraction method. Furthermore, we implement a parallelized LSQR algorithm based on graphics processing unit programming in the Compute Unified Device Architecture to accelerate the computational processing. Experimental results illustrate that the proposed extraction method can greatly reduce the computational cost and memory use of the deconvolution method and, consequently, increase its efficiency and practicability. In addition, the proposed extraction method has a stronger noise tolerance than other methods, such as the boxcar (aperture) extraction and profile extraction methods. Finally, we present an analysis of the sensitivity of the extraction results to the radius and full width at half-maximum of the 2D PSF.

  14. Molecular Engineering of Liquid Crystal Polymers by Living Polymerization. 17. Characterization of Poly(10-((4-Cyano-4’-Biphenyl)oxy) decanyl Vinyl Ether)s by 1-D and 2-D H-NMR Spectroscopy

    DTIC Science & Technology

    1991-10-30

    Spectroscopy by Virril Percec and Myongsoo Lee Department of Macromolecular Science Case Western Reserve University Cleveland, OH 44106-2699 and Peter L ...AUTHOrZ(S) Virgil Percec, Myongsoo Lee, Peter L . Rinaldi and Vincent E. Litman l3a TYPE OF REPORT 1131) TIME COVERED 14. DATE OF REPORT (Year. Afot? Dy I...with CF3SO 3 H/S(CH 3)2 in CH2Cl2 at 0OC and termninated by ammoniacal methanol, by 1 -D and 2-D (COSY) 300 MHz IH-NMR spectroscopy is presented. The

  15. Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy.

    PubMed

    Bixler, Joel N; Cone, Michael T; Hokr, Brett H; Mason, John D; Figueroa, Eleonora; Fry, Edward S; Yakovlev, Vladislav V; Scully, Marlan O

    2014-05-20

    Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment.

  16. Determination of Yttrium in High Density Silicon Nitride by Emission and X-Ray Fluorescence Spectroscopy.

    DTIC Science & Technology

    1981-08-01

    AD-AI07 596 ARMY MATERIALS AND MECHANICS RESEARCH CENTER WATERTOWN MA F/S 7/4 DETERMINATION OF YTTRIUM IN HIGH DENSITY SILICON NITRIDE BY EMI-ETCIU...AUG Al B H STRAUSS. UNCLASSIFIED AMMRC-TR-Al-39 N AMMRC TR 81-39 A ~LEVEL ’ t’- .- DETERMINATION OF YTTRIUM IN 1 HIGH DENSITY SILICON NITRIDE BY...DETERMINATION OF YTTRIUM IN HIGH DENSITY SILICON NITRIDE BY EMISSION AND X-RAY Final Report FLUORESCENCE SPECTROSCOPY 6 PERFORMING ORG. REPORT NUMBER 7. AUTHOR

  17. Time-resolved fluorescence spectroscopy for clinical diagnosis of actinic cheilitis.

    PubMed

    Cosci, Alessandro; Nogueira, Marcelo Saito; Pratavieira, Sebastião; Takahama, Ademar; Azevedo, Rebeca de Souza; Kurachi, Cristina

    2016-10-01

    Actinic cheilitis is a potentially malignant disorder of the lips. Its first cause is believed to be UV sun radiation. The lesion is highly heterogeneous, making the choice of area to be biopsied difficult. This study exploits the capabilities of time-resolved fluorescence spectroscopy for the identification of the most representative area to be biopsied. A preliminary study was performed on fourteen patients. A classification algorithm was used on data acquired on nine different biopsies. The algorithm discriminated between absent, mild, and moderate dysplasia with a sensitivity of 92.9%, 90.0%, and 80.0%, respectively. The false positive rate for healthy tissue (specificity) was 88.8%.

  18. Detection of citrus canker and Huanglongbing using fluorescence imaging spectroscopy and support vector machine technique.

    PubMed

    Wetterich, Caio Bruno; Felipe de Oliveira Neves, Ruan; Belasque, José; Marcassa, Luis Gustavo

    2016-01-10

    Citrus canker and Huanglongbing (HLB) are citrus diseases that represent a serious threat to the citrus production worldwide and may cause large economic losses. In this work, we combined fluorescence imaging spectroscopy (FIS) and a machine learning technique to discriminate between these diseases and other ordinary citrus conditions that may be present at citrus orchards, such as citrus scab and zinc deficiency. Our classification results are highly accurate when discriminating citrus canker from citrus scab (97.8%), and HLB from zinc deficiency (95%). These results show that it is possible to accurately identify citrus diseases that present similar symptoms.

  19. Hierarchical organization of the plasma membrane: investigations by single-molecule tracking vs. fluorescence correlation spectroscopy.

    PubMed

    Kusumi, Akihiro; Shirai, Yuki M; Koyama-Honda, Ikuko; Suzuki, Kenichi G N; Fujiwara, Takahiro K

    2010-05-03

    Single-molecule tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a number of unprecedented observations, thus fostering a new basic understanding of molecular diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, containing diverse structures on nano-meso-scales (2-200 nm) with a variety of lifetimes, where certain membrane molecules stay together for limited durations. Molecular interactions occur in the time-dependent inhomogeneous two-dimensional liquid of the plasma membrane, which might be a key for plasma membrane functions.

  20. Interactions of hypericin with a model mutagen - Acridine orange analyzed by light absorption and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Pietrzak, Monika; Szabelski, Mariusz; Kasparek, Adam; Wieczorek, Zbigniew

    2017-02-01

    The present study was designed to estimate the ability of hypericin to interact with a model mutagen - acridine orange. The hetero-association of hypericin and acridine orange was investigated with absorption and fluorescence spectroscopy methods in aqueous solution of DMSO. The data indicate that hypericin forms complexes with acridine orange and that the association constants are relatively high and depend on DMSO concentration. The absorption spectra of the hypericin - acridine orange complexes were examined as well. Owing to its ability to interact with flat aromatic compounds, hypericin may potentially be used as an interceptor molecule.

  1. On the measurement of particle number and mobility in nonideal solutions by fluorescence correlation spectroscopy.

    PubMed Central

    Abney, J R; Scalettar, B A; Hackenbrock, C R

    1990-01-01

    Interparticle interactions are incorporated into the theoretical description of the initial amplitude, G(0), of the normalized fluorescence correlation spectroscopy autocorrelation function. Measurements of particle number, aggregate size, and interaction-dependent diffusion are then analyzed in the context of this generalized theory. It is shown that the neglect of interactions can introduce order-of-magnitude errors into estimates of particle number and aggregate size. It is also shown that measurement of G(0) provides an essentially unique method for testing the validity of theories of interaction-dependent membrane protein diffusion. PMID:2383634